Method of producing secreted receptor analogs and biologically active peptide dimers

ABSTRACT

Methods for producing secreted receptor analogs and biologically active peptide dimers are disclosed. The methods for producing secreted receptor analogs and biologically active peptide dimers utilize a DNA sequence encoding a receptor analog or a peptide requiring dimerization for biological activity joined to a dimerizing protein. The receptor analog includes a ligand-binding domain. Polypeptides comprising essentially the extracellular domain of a human PDGF receptor fused to dimerizing proteins, the portion being capable of binding human PDGF or an isoform thereof, are also disclosed. The polypeptides may be used within methods for determining the presence of and for purifying human PDGF or isoforms thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.146,877, filed Jan. 22, 1988, which application is now abandoned.

DESCRIPTION

1. Technical Field

The present invention is generally directed toward the expression ofproteins, and more specifically, toward the expression of growth factorreceptor analogs and biologically active peptide dimers.

2. Background of the Invention

In higher eucaryotic cells, the interaction between receptors andligands (e.g., hormones) is of central importance in the transmission ofand response to a variety of extracellular signals. It is generallyaccepted that hormones and growth factors elicit their biologicalfunctions by binding to specific recognition sites (receptors) in theplasma membranes of their target cells. Upon ligand binding, a receptorundergoes a conformational change, triggering secondary cellularresponses that result in the activation or inhibition of intracellularprocesses. The stimulation or blockade of such an interaction bypharmacological means has important therapeutic implications for a widevariety of illnesses.

Ligands fall into two classes: those that have stimulatory activity,termed agonists; and those that block the effects elicited by theoriginal ligands, termed antagonists. The discovery of agonists thatdiffer in structure and composition from the original ligand may bemedically useful. In particular, agonists that are smaller than theoriginal ligand may be especially useful. The bioavailability of thesesmaller agonists may be greater than that of the original ligand. Thismay be of particular importance for topical applications and forinstances when diffusion of the agonist to its target sites is inhibitedby poor circulation. Agonists may also have slightly different spectraof biological activity and/or different potencies, allowing them to beused in very specific situations. Agonists that are smaller andchemically simpler than the native ligand may be produced in greaterquantity and at lower cost. The identification of antagonists whichspecifically block, for example, growth factor receptors has importantpharmaceutical applications. Antagonists that block receptors againstthe action of endogenous, native ligand may be used as therapeuticagents for conditions including atherosclerosis, autocrine tumors,fibroplasia and keloid formation.

The discovery of new ligands that may be used in pharmaceuticalapplications has centered around designing compounds by chemicalmodification, complete synthesis, and screening potential ligands bycomplex and costly screening procedures. The process of designing a newligand usually begins with the alteration of the structure of theoriginal effector molecule. If the original effector molecule is knownto be chemically simple, for example, a catecholamine or prostaglandin,the task is relatively straightforward. However, if the ligand isstructurally complex, for example, a peptide hormone or a growth factor,finding a molecule which is functionally equivalent to the originalligand becomes extremely difficult.

Currently, potential ligands are screened using radioligand bindingmethods (Lefkowitz et al., Biochem. Biophys. Res. Comm. 60: 703-709,1974; Aurbach et al., Science 186: 1223-1225, 1974; Atlas et al. Proc.Natl. Acad. Sci. USA 71: 4246-4248, (1974). Potential ligands can bedirectly assayed by binding the radiolabeled compounds to responsivecells, to the membrane fractions of disrupted cells, or to solubilizedreceptors. Alternatively, potential ligands may be screened by theirability to compete with a known labeled ligand for cell surfacereceptors.

The success of these procedures depends on the availability ofreproducibly high quality preparations of membrane fractions or receptormolecules, as well as the isolation of responsive cell lines. Thepreparation of membrane fractions and soluble receptor moleculesinvolves extensive manipulations and complex purification steps. Theisolation of membrane fractions requires gentle manipulation of thepreparation, a procedure which does not lend itself to commercialproduction. It is very difficult to maintain high biological activityand biochemical purity of receptors when they are purified by classicalprotein chemistry methods. Receptors, being integral membrane proteins,require cumbersome purification procedures, which include the use ofdetergents and other solvents that interfere with their biologicalactivity. The use of these membrane preparations in ligand bindingassays typically results in low reproducibility due to the variabilityof the membrane preparations.

As noted above, ligand binding assays require the isolation ofresponsive cell lines. Often, only a limited subset of cells isresponsive to a particular agent, and such cells may be responsive onlyunder certain conditions. In addition, these cells may be difficult togrow in culture or may possess a low number of receptors. Currentlyavailable cell types responsive to platelet-derived growth factor(PDGF), for example, contain only a low number (up to 4×10⁵ ; seeBowen-Pope and Ross, J. Biol. Chem. 5161-5171, 1982) of receptors percell, thus requiring large numbers of cells to assay PDGF analogs orantagonists.

Presently, only a few naturally-occurring secreted receptors, forexample, the interleukin-2 receptor (IL-2-R) have been identified. Rubinet al. (J. Immun. 135: 3172-3177, 1985) have reported the release oflarge quantities of IL-2-R into the culture medium of activated T-celllines. Bailon et al. (Bio/Technology 1195-1198, 1987) have reported theuse of a matrix-bound interleukin-2 receptor (IL-2-R) to purifyrecombinant interleukin-2.

Three other receptors have been secreted from mammalian cells. Theinsulin receptor (Ellis et al., J. Cell Biol. 150: 14a, 1987), the HIV-1envelope glycoprotein cellular receptor CD4 (Smith et al., Science 238:1704-1707, 1987) and the epidermal growth factor (EGF) receptor (Livnehet al., J. Biol. Chem. 261: 12490-12497, 1986) have been secreted frommammalian cells using truncated cDNAs that encode portions of theextracellular domains.

Naturally-occurring secreted receptors have not been widely identified,and there have been only a limited number of reports of secretedrecombinant ligand-binding receptors. Secreted ligand-binding receptorsmay be used in a variety of assays, which include assays to determinethe presence of ligand in biological fluids and assays to screen forpotential agonists and antagonists. Current methods for ligand screeningand ligand/receptor binding assays have been limited to those usingpreparations of whole cells or cell membranes for as a source forreceptor molecules. The low reproducibility and high cost of producingsuch preparations does not lend itself to commercial production. Thereis therefore a need in the art for a method of producing secretedreceptors. There is a further need in the art for an assay system thatpermits high volume screening of compounds that may act on highereucaryotic cells via specific surface receptors. This assay systemshould be rapid, inexpensive and adaptable to high volume screening. Thepresent invention discloses such a method and assay system, and furtherprovides other related advantages.

DISCLOSURE OF INVENTION

Briefly stated, the present invention discloses methods for producingsecreted receptor analogs which including ligand-binding receptoranalogs and secreted platelet-derived growth factor receptor (PDGF-R)analogs. In addition, the present invention discloses methods forproducing secreted peptide dimers.

Within one aspect of the invention a method for producing a secretedPDGF-R analog is disclosed, comprising (a) introducing into a eukaryotichost cell a DNA construct capable of directing the expression andsecretion of a PDGF receptor analog, the DNA construct comprising atranscriptional promoter operatively linked to at least one secretorysignal sequence followed downstream in proper reading frame by a DNAsequence encoding at least a portion of the extracellular domain of aPDGF-R, the portion including a ligand-binding domain; (b) growing thehost cell in an appropriate growth medium; and (c) isolating the PDGF-Ranalogs from the host cell.

Within one embodiment, the dimerizing protein is yeast invertase. Withinanother embodiment, the dimerizing protein is at least a portion of animmunoglobulin light chain. Within another embodiment, the dimerizingprotein is at least a portion of an immunoglobulin heavy chain.

Within one embodiment of the present invention, a PDGF-R analogcomprising the amino acid sequence of FIG. 1 from isoleucine, number 29to methionine, number 441 is secreted. Within another embodiment aPDGF-R analog comprising the amino acid sequence of FIG. 1 fromisoleucine, number 29 to lysine, number 531 is secreted.

Yet another aspect of the present invention discloses a method forproducing a secreted, biologically active peptide dimer. The methodgenerally comprises a) introducing into a eukaryotic host cell a DNAconstruct capable of directing the expression and secretion of a peptiderequiring dimerization for biological activity, the DNA constructcomprising a transcriptional promoter operatively linked to at least onesecretory signal sequence followed downstream by and in proper readingframe with a DNA sequence encoding a peptide requiring dimerization forbiological activity joined to a dimerizing protein; (b) growing the hostcell in an appropriate growth medium under physiological conditions toallow the dimerization and secretion of the peptide; and (c) isolatingthe biologically active peptide dimer from the host cell.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active peptide dimer, comprising (a)introducing into a eukaryotic host cell a first DNA construct comprisinga transcriptional promoter operatively linked to a first secretorysignal sequence followed downstream by and in proper reading frame witha first DNA sequence encoding a peptide requiring dimerization forbiological activity joined to an immunoglobulin light chain constantregion; (b) introducing into the host cell a second DNA constructcomprising a transcriptional promoter operatively linked to a secondsecretory signal sequence followed downstream by and in proper readingframe with a second DNA sequence encoding at least one immunoglobulinheavy chain constant region domain selected from the group consisting ofC_(H) 1, C_(H) 2, C_(H) 3, and C_(H) 4; (c) growing the host cell in anappropriate growth medium under physiological conditions to allow thedimerization and secretion of the biologically active peptide dimer; and(d) isolating the biologically active peptide dimer from the host cell.In one embodiment, the second DNA sequence further encodes animmunoglobulin heavy chain hinge region wherein the hinge region isjoined to at least one heavy chain constant region domain. In apreferred embodiment, the second DNA sequence further encodes animmunoglobulin variable region joined upstream of and in proper readingframe with at least one immunoglobulin heavy chain constant region.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active peptide dimer, comprising (a)introducing into a eukaryotic host cell a first DNA construct comprisinga transcriptional promoter operatively linked to a first secretorysignal sequence followed downstream by and in proper reading frame witha first DNA sequence encoding a peptide requiring dimerization forbiological activity joined to at least one immunoglobulin heavy chainconstant region domain selected from the group consisting of C_(H) 1,C_(H) 2, C_(H) 3, and C_(H) 4; (b) introducing into the host cell asecond DNA construct comprising a transcriptional promoter operativelylinked to a second secretory signal sequence followed downstream by andin proper reading frame with a second DNA sequence encoding animmunoglobulin light chain constant region; (c) growing the host cell inan appropriate growth medium under physiological conditions to allow thedimerization and secretion of the biologically active peptide dimer; and(d) isolating the biologically active peptide dimer from the host cell.In one embodiment, the first DNA sequence further encodes animmunoglobulin heavy chain hinge region wherein the hinge region isjoined to at least one heavy chain constant region domain. In apreferred embodiment, the second DNA sequence further encodes animmunoglobulin variable region joined upstream of and in proper readingframe with an immunoglobulin light chain constant region.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active peptide dimer, comprising (a)introducing into a eukaryotic host cell a DNA construct comprising atranscriptional promoter operatively linked to a secretory signalsequence followed downstream by and in proper reading frame with a DNAsequence encoding a peptide requiring dimerization for biologicalactivity joined to at least one immunoglobulin heavy chain constantregion domain selected from the group consisting of C_(H) 1, C_(H) 2,C_(H) 3, and C_(H) 4; (b) growing the host cell in an appropriate growthmedium under physiological conditions to allow the dimerization andsecretion of the biologically active peptide dimer; and (c) isolatingthe biologically active peptide dimer from the host cell. In oneembodiment, the DNA sequence further encodes an immunoglobulin heavychain hinge region wherein the hinge region is joined to at least oneheavy chain constant region domain.

In another aspect of the invention, a method is disclosed for producinga secreted, biologically active peptide dimer, comprising (a)introducing into a eukaryotic host cell a first DNA construct comprisinga transcriptional promoter operatively linked to a first secretorysignal sequence followed downstream in by and in proper reading framewith a first DNA sequence encoding a first polypeptide chain of apeptide dimer joined to at least one immunoglobulin heavy chain constantregion domain, selected from the group consisting of C_(H) 1, C_(H) 2,C_(H) 3, and C_(H) 4; (b) introducing into the host cell a second DNAconstruct comprising a transcriptional promoter operatively linked to asecond secretory signal sequence followed downstream by and in properreading frame with a second DNA sequence encoding a second polypeptidechain of a peptide dimer joined to an immunoglobulin light chainconstant region domain; (c) growing the host cell in an appropriategrowth medium under physiological conditions to allow the dimerizationand secretion of the biologically active peptide dimer; and (d)isolating the biologically acitve peptide dimer from the host cell. Inone embodiment the first DNA sequence further encodes an immunoglobulinheavy chain hinge region domain wherein the hinge region is joined to atleast one immunoglobulin heavy chain constant region domain. In apreferred embodiment, the second DNA sequence further encodes animmunoglobulin variable region joined upstream of and in proper readingframe with an immunoglobulin light chain constant region.

In yet another aspect of the invention, a method is disclosed forproducing a secreted, ligand-binding receptor analog, comprising (a)introducing into a eukaryotic host cell a first DNA construct comprisinga transcriptional promoter operatively linked to a first secretorysignal sequence followed downstream by and in proper reading frame witha first DNA sequence encoding a polypeptide chain of a ligand-bindingreceptor analog joined to at least an immunoglobulin light chainconstant region; (b) introducing into the host cell a second DNAconstruct comprising a transcriptional promoter operatively linked to asecond secretory signal sequence followed downstream by and in properreading frame with a second DNA sequence encoding at least oneimmunoglobulin heavy chain constant region domain, selected from thegroup consisting of C_(H) 1, C_(H) 2, C_(H) 3, and C_(H) 4; (c) growingthe host cell in an appropriate growth medium under physiologicalconditions to allow the dimerization and secretion of the ligand-bindingreceptor analog; and (d) isolating the ligand-binding receptor analogfrom the host cell. In one embodiment, the second DNA sequence furtherencodes an immunoglobulin heavy chain hinge region wherein the hingeregion is joined to at least one heavy chain constant region domain. Ina preferred embodiment, the second DNA sequence further encodes animmunoglobulin variable region joined upstream of and in proper readingframe with at least one immunoglobulin heavy chain constant region.

In another aspect of the invention, a method is disclosed for producinga secreted, ligand-binding receptor analog, comprising (a) introducinginto a eukaryotic host cell a DNA construct comprising a transcriptionalpromoter operatively linked to a secretory signal sequence followeddownstream by and in proper reading frame with a DNA sequence encoding apolypeptide chain of a ligand-binding receptor analog joined to at leastone immunoglobulin heavy chain constant region domain, selected from thegroup C_(H) 1, C_(H) 2, C_(H) 3, and C_(H) 4; (b) growing the host cellin an appropriate growth medium under physiological conditions to allowthe dimerization and secretion of the ligand-binding receptor analog;and (c) isolating the ligand-binding receptor analog from the host cell.In one embodiment, the DNA sequence further encodes an immunoglobulinheavy chain hinge region wherein the hinge region is joined to at leastone heavy chain constant region domain.

In another aspect of the invention, a method is disclosed for producinga secreted, ligand-binding receptor analog, comprising (a) introducinginto a eukaryotic host cell a first DNA construct comprising atranscriptional promoter operatively linked to a first secretory signalsequence followed downstream in proper reading frame by a first DNAsequence encoding a polypeptide chain of a ligand-binding receptoranalog joined to at least one immunoglobulin heavy chain constant regiondomain, selected from the group C_(H) 1, C_(H) 2, C_(H) 3, and C_(H) 4;(b) introducing into the host cell a second DNA construct comprising atranscriptional promoter operatively linked to a second secretory signalsequence followed downstream by and in proper reading frame with asecond DNA sequence encoding at least an immunoglobulin light chainconstant region; (c) growing the host cell in an appropriate growthmedium under physiological conditions to allow the dimerization andsecretion of the ligand-binding receptor analog; and (d) isolating theligand-binding receptor analog from the host cell. In one embodiment,the first DNA sequence further encodes an immunoglobulin heavy chainhinge region wherein the hinge region is joined to at least one heavychain constant region domain. In a preferred embodiment, the second DNAsequence further encodes an immunoglobulin variable region joinedupstream of and in proper reading frame with an immunoglobulin lightchain constant region.

In another aspect of the invention, a method is disclosed for producinga secreted, ligand-binding receptor analog, comprising (a) introducinginto a eukaryotic host cell a first DNA construct comprising atranscriptional promoter operatively linked to a first secretory signalsequence followed downstream in proper reading frame by a first DNAsequence encoding a first polypeptide chain of a ligand-binding receptoranalog joined to at least one immunoglobulin heavy chain constant regiondomain, selected from the group C_(H) 1, C_(H) 2, C_(H) 3, and C_(H) 4;(b) introducing into the host cell a second DNA construct comprising atranscriptional promoter operatively linked to a second secretory signalsequence followed downstream by and in proper reading frame with asecond DNA sequence encoding a second polypeptide chain of a ligandbinding receptor analog joined to an immunoglobulin light chain constantregion domain. In one embodiment the first DNA sequence further encodesan immunoglobulin heavy chain hinge region domain wherein the hingeregion is joined to at least one immunoglobulin heavy chain constantregion domain.

Host cells for use in the present invention include cultured mammaliancells and fungal cells. In a preferred embodiment strains of the yeastSaccharomyces cerevisiae are used as host cells. Within anotherpreferred embodiment cultured rodent myeloma cells are used as hostcells.

Within one embodiment of the present invention, a PDGF-R analogcomprises the amino acid sequence of FIG. 1 from isoleucine, number 29to methionine, number 441. Within another embodiment a PDGF-R analogcomprises the amino acid sequence of FIG. 1 from isoleucine, number 29to lysine, number 531. PDGF-R analogs produced by the above-disclosedmethods may be used, for instance, within a method for determining thepresence of human PDGF or an isoform thereof in a biological sample.

A method for determining the presence of human PDGF or an isoformthereof in a biological sample is disclosed and comprises (a) incubatinga polypeptide comprising a human PDGF receptor analog fused to adimerizing protein with a biological sample suspected of containinghuman PDGF or an isoform thereof under conditions that allow theformation of receptor/ligand complexes; and (b) detecting the presenceof receptor/ligand complexes, and therefrom determining the presence ofhuman PDGF or an isoform thereof. Suitable biological samples in thisregard include blood, urine, plasma, serum, platelet and other celllysates, platelet releasates, cell suspensions, cell-conditioned culturemedia, and chemically or physically separated portions thereof.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 1C illustrate the nucleotide sequence of the PDGF receptorcDNA and the derived amino acid sequence of the primary translationproduct. Numbers above the lines refer to the nucleotide sequence;numbers below the lines refer to the amino acid sequence.

FIG. 2 illustrates the construction of pBTL10, pBTL11 and pBTL12.

FIG. 3 illustrates the construction of pCBS22.

FIG. 4 illustrates the construction of pBTL13 and pBTL14.

FIG. 5 illustrates the construction of pBTL15.

FIG. 6 illustrates the construction of pBTL22 and pBTL26.

FIG. 7 illustrates the construction of pSDL114. Symbols used are S.S.,signal sequence, C_(k), immunoglobulin light chain constant regionsequence; μ prom, μ promoter, μ enh; μ enhancer.

FIG. 8 illustrates the construction of pSDLB113. Symbols used are S.S.,signal sequence; C_(H) 1, C_(H) 2, C_(H) 3, immunoglobulin heavy chainconstant region domain sequences; H, immunoglobulin heavy chain hingeregion sequence; M, immunoglobulin membrane anchor sequences; C.sub.γ1M, immunoglobulin heavy chain constant region and membrane anchorsequences.

FIG. 9 illustrates the constructions pBTL115, pBTL114, pφ5V_(H)HuC.sub.γ 1M-neo, plCφ5V.sub.κ HuC.sub.κ -neo. Symbols used are setforth in FIGS. 7 and 8, and also include L_(H), mouse immunoglobulinheavy chain signal sequence; V_(H), mouse immunoglobulin heavy chainvariable region sequence; E, mouse immunoglobulin heavy chain enhancersequence; L.sub.κ, mouse immunoglobulin light chain signal sequence;φ5V.sub.κ, mouse immunoglobulin light chain variable region sequence;Neo^(R), E. coli neomycin resistance gene.

FIG. 10 illustrates the constructions Zem229R, pφ5V_(H) Fab-neo andpWKI. Symbols used are set forth in FIG. 10.

BEST MODE FOR CARRYING OUT THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

DNA Construct: A DNA molecule, or a clone of such a molecule, eithersingle- or double-stranded that has been modified through humanintervention to contain segments of DNA combined and juxtaposed in amanner that as a whole would not otherwise exist in nature.

Secretory Signal Sequence: A DNA sequence encoding a secretory peptide.A secretory peptide is an amino acid sequence that acts to direct thesecretion of a mature polypeptide or protein from a cell. Secretorypeptides are characterized by a core of hydrophobic amino acids and aretypically (but not exclusively) found at the amino termini of newlysythesized proteins. Very often the secretory peptide is cleaved fromthe mature protein during secretion. Such secretory peptides containprocessing sites that allow cleavage of the signal peptides from themature proteins as it passes through the secretory pathway. Processingsites may be encoded within the signal peptide or may be added to thesignal peptide by, for example, vitro mutagenesis. Certain secretorypeptides may be used in concert to direct the secretion of polypeptidesand proteins. One such secretory peptide that may be used in combinationwith other secretory peptides is the third domain of the yeast Barrierprotein.

Receptor Analog: A portion of a receptor capable of binding ligand,anti-receptor antibodies, analogs or antagonists. The amino acidsequence of the receptor analog may contain additions, substitutions ordeletions as compared to the native receptor sequence. A receptor analogmay be, for example, the ligand-binding domain of a receptor joined toanother protein. Platelet-derived growth factor receptor (PDGF-R)analogs may, for example, comprise a portion of a PDGF receptor capableof binding anti-PDGF receptor antibodies, PDGF, PDGF isoforms, PDGFanalogs, or PDGF antagonists.

Dimerizing Protein: A polypeptide chain having affinity for a secondpolypeptide chain, such that the two chains associate underphysiological conditions to form a dimer. The second polypeptide chainmay be the same or a different chain.

Biological activity: A function or set of activities performed by amolecule in a biological context (i.e., in an organism or an in vitrofacsimile thereof). Biological activities may include the induction ofextracellular matrix secretion from responsive cell lines, the inductionof hormone secretion, the induction of chemotaxis, the induction ofmitogenesis, the induction of differentiation, or the inhibition of celldivision of responsive cells. A recombinant protein or peptide isconsidered to be biologically active if it exhibits one or morebiological activities of its native counterpart.

Ligand: A molecule, other than an antibody or an immunoglobulin, capableof being bound by the ligand-binding domain of a receptor. The moleculemay be chemically synthesized or may occur in nature.

Joined: Two or more DNA coding sequences are said to be joined when, asa result of in-frame fusions between the DNA coding sequences or as aresult of the removal of intervening sequences by normal cellularprocessing, the DNA coding sequences are translated into a polypeptidefusion.

As noted above, the present invention provides methods for producingbiologically active peptide dimers and secreted receptor analogs, whichinclude ligand-binding receptor analogs and PDGF receptor analogs.Secreted receptor analogs may be used to screen for new compounds thatact as agonists or antagonists when interacting with cells containingmembrane-bound receptors. In addition, the methods of the presentinvention provide peptides of therapeutic value that are biologicallyactive only as dimers. Moreover, the present invention provides methodsof producing peptide dimers that are biologically active only asnon-covalently associated dimers. Secreted, biologically active dimersthat may be produced using the present invention include nerve growthfactor, colony stimulating factor-1, factor XIII, and transforminggrowth factor β.

Ligand-binding receptor analogs that may be used in the presentinvention include the ligand-binding domains of the epidermal growthfactor receptor (EGF-R) and the insulin receptor. As used herein, aligand-binding domain is that portion of the receptor that is involvedin binding ligand and is generally a portion or essentially all of theextracellular domain that extends from the plasma membrane into theextracellular space. The ligand-binding domain of the EGF-R, forexample, resides in the extracellular domain. EGF-R dimers have beenfound to exhibit higher ligand-binding affinity than EGF-R monomers(Boni-Schnetzler and Pilch, Proc. Natl. Acad. Sci. USA 84:7832-7836,1987). The insulin receptor (Ullrich et al., Nature 313:756-761, 1985)requires dimerization for biological activity.

Another example of a receptor that may be secreted from a host cell is aplatelet-derived growth factor receptor (PDGF-R). Two classes ofPDGF-Rs, which recognized different isoforms of PDGF, have beenidentified. (PDGF is a disulfide-bonded, two-chain molecule, which ismade up of an A chain and a B chain. These chains may be combined as ABheterodimers, AA homodimers or BB homodimers. These dimeric molecules Lare referred to herein as "isoforms".) The β-receptor, which recognizesonly the BB isoform of PDGF (PDGF-BB), has been described(Claesson-Welsh et al., Mol. Cell. Biol. 8:3476-3486, 1988; Gronwald etal., Proc. Natl. Acad. Sci. USA 85:3435-3439, 1988). The α-receptor,which recognizes all three PDGF isoforms (PDGF-AA, PDGF-AB and PDGF-BB),has been described by Matsui et al. (Science 243:800-804, 1989). Theprimary translation products of these receptors indicated that eachincludes an extracellular domain implicated in the ligand-bindingprocess, a transmembrane domain, and a cytoplasmic domain containing atyrosine kinase activity. Matsui et al. (ibid.) have predicted theligand-binding domain of the α-PDGF receptor to include amino acids25-500 of their reported α-receptor amino acid sequence. Gronwald et al.(ibid.) have predicted the ligand-binding domain of the β-PDGF receptorto include amino acids 29-531 of their published amino acid sequence.

The present invention provides a standardized assay system, notpreviously available in the art, for determining the presence of PDGF,PDGF isoforms, PDGF agonists or PDGF antagonists using a secreted PDGFreceptor analogs. Such an assay system will typically involve combiningthe secreted PDGF receptor analog with a biological sample underphysiological conditions which permit the formation of receptor-ligandcomplexes, followed by detecting the presence of the receptor-ligandcomplexes. Detection may be achieved through the use of a label attachedto the PDGF receptor analog or through the use of a labeled antibodywhich is reactive with the receptor analog or the ligand. A wide varietyof labels may be utilized, such as radionuclides, fluorophores, enzymesand luminescers. Receptor-ligand complexes may also be detectedvisually, i.e., in immunoprecipitation assays which do not require theuse of a label. This assay system provides secreted PDGF receptoranalogs that may be utilized in a variety of screening assays for, forexample, screening for analogs of PDGF. The present invention alsoprovides a methods for measuring the level of PDGF and PDGF isoforms inbiological fluids.

As noted above, the present invention provides methods for producingpeptide dimers that require dimerization for biological activity orenhancement of biological activity. Peptides requiring dimerization ofbiological activity include, in addition to certain receptors nervegrowth factor, colony-stimulating factor-1 (CSF-1), transforming growthfactor β (TGF-β), PDGF, and factor XIII. Nerve growth factor is anon-covalently linked dimer (Harper et al., J. Biol. Chem. 257:8541-8548, 1982). CSF-1, which specifically stimulates the proliferationand differentiation of cells of mononuclear phagocytic lineage, is adisulfide-bonded homodimer (Retternmier et al., Mol. Cell. Biol. 7:2378-2387, 1987). TGF-β is biologically active as a disulfide-bondeddimer (Assoian et al., J. Biol. Chem. 258:7155-7160, 1983). Factor XIIIis a plasma protein that exists as a two chain homodimer in itsactivated form (Ichinose et al., Biochem. 25: 6900-6906, 1986). PDGF, asnoted above, is a disulfide-bonded, two chain molecule (Murray et al.,U.S. Pat. No. 4,766,073).

The present invention provides methods by which receptor analogs,including ligand-binding receptor analogs and PDGF-R analogs, requiringdimerization for activity may be secreted from host cells. The methodsdescribed herein are particularly advantageous in that they allow theproduction of large quantities of purified receptors. The receptors maybe used in assays for the screening of potential ligands, in assays forbinding studies, as imaging agents, and as agonists and antagonistswithin therapeutic agents.

A DNA sequence encoding a human PDGF receptor may be isolated as a cDNAusing techniques known in the art (see, for example, Okayama and Berg,Mol. Cell. Biol. 161-170, 1982; Mol. Cell. Biol. 3: 280-289, 1983). ADNA molecule encoding a human PDGF receptor may be isolated from alibrary of human genomic or cDNA sequences. Such libraries may beprepared by standard procedures, such as those disclosed by Gubler andHoffman (Gene 25: 263=269, 1983). It is preferred that the molecule is acDNA molecule because cDNA lacks introns and is therefore more suited tomanipulation and expression in transfected or transformed cells. Sourcesof mRNA for use in the preparation of a cDNA library include the MG-63human osteosarcoma cell line (available from ATCC under accession numberCRL 1427), diploid human dermal fibroblasts and human embryo fibroblastand brain cells (Matsui et al., ibid.). A cDNA encoding a β-PDGF-R hasbeen cloned from a diploid human dermal fibroblast cDNA library usingoligonucleotide probes complementary to sequences of the mouse PDGFreceptor (Gronwald et al., ibid.). An α-PDGF-R cDNA has been isolated byMatsui et al. (ibid.) from human embryo fibroblast and brain cells.Alternatively, a cDNA encoding an α-PDGF-R may be isolated from alibrary prepared from MG-63 human osteosarcoma cells using a cDNA probecontaining sequences encoding a cytoplasmic domain of the β-PDGF-R.Partial cDNA clones (fragments) can be extended by re-screening of thelibrary with the cloned cDNA fragment until the full sequence isobtained. In a preferred embodiment, a DNA sequence encoding a PDGFreceptor analog consisting essentially of the extracellular domain of aPDGF receptor is used, although smaller DNA sequences encoding portionsof at least 40% amino acids of the extracellular domain may be used.

DNA sequences encoding EGF-R (Ullrich et al., Nature 304: 418-425,1984), the insulin receptor (Ullrich et al., Nature 756-761, 1985),nerve growth factor (Ullrich et al. Nature 303: 821-825, 1983), colonystimulating factor-1 (Rettenmier et al., ibid.), transforming growthfactor β (Derynck et al., Nature 316: 701-705, 1985), PDGF (Murray etal., ibid.), and factor XIII (Ichinose et al., ibid.) may also be usedwithin the present invention.

To direct peptides requiring dimerization for biological activity orreceptor analogs into the secretory pathway of the host cell, at leastone secretory signal sequence is used in conjunction with the DNAsequence of interest. Preferred secretory signals include the alphafactor signal sequence (pre-pro sequence) (Kurjan and Herkowitz, Cell30: 933-943, 1982; Kurjan et al., U.S. Pat. No. 4,546,082; Brake, EP116,201, 1983), the PHO5 signal sequence (Beck et al., WO 86/00637), theBARI secretory signal sequence (MacKay et al., U.S. Pat. No. 4,613,572;MacKay, WO 87/002670), immunoglobulin V_(H) signal sequences (Loh etal., Cell: 85-93, 1983; Watson Nuc. Acids. Res. 12: 5145-5164, 1984) andimmunoglobulin V.sub.κ signal sequences (Watson, ibid.). Particularlypreferred signal sequences are the SUC2 signal sequence (Carlson et al.,Mol. Cell. Biol. 439-447, 1983) and PDGF receptor signal sequences.Alternatively, secretory signal sequences may be synthesized accordingto the rules established, for example, by von Heinje (Eur. J. Biochem.133: 17-21, 1983; J. Mol. Biol. 99-105 1985; Nuc. Acids. Res. 14:4683-3690, 1986).

Secretory signal sequences may be used singly or may be combined. Forexample, a first secretory signal sequence may be used singly orcombined with a sequence encoding the third domain of Barrier (describedin co-pending commonly assigned U.S. patent application Ser. No.104,316, now abandoned, which is incorporated by reference herein in itsentirety). The third domain of Barrier may be positioned in properreading frame 3' of the DNA sequence of interest or 5' to the DNAsequence and in proper reading frame with both the secretory signalsequence and the DNA sequence of interest.

In one embodiment of the present invention, a sequence encoding adimerizing protein is joined to a sequence encoding a peptide dimer or areceptor analog, and this fused sequence is joined in proper readingframe to a secretory signal sequence. As shown herein, the presentinvention utilizes such an arrangement to drive the association of thepeptide or receptor analog to form a biologically active molecule uponsecretion. Suitable dimerizing proteins include the S. cerevisiaerepressible acid phosphatase (Mizunaga et al., J. Biochem. (Tokyo) 103:321-326, 1988), the S. cerevisiae type 1 killer preprotoxin (Sturley etal., EMBO J. 5: 3381-3390, 1986), the S. calsberoensis alphagalactosidase melibiase (Sumner-Smith et al., Gene 36: 333-340, 1985),the S. cerevisiae invertase gene (Carlson et al., Mol. Cell. Biol. 5:439-447, 1983), the Neurospora crassa ornithine decarboxylase (Digangiet al., J. Biol. Chem. 262: 7889-7893, 1987), immunoglobulin heavy chainhinge regions (Takahashi et al., Cell 29: 671-679, 1982), and otherdimerizing immunoglobulin sequences. In a preferred embodiment, S.cerevisiae invertase is used to drive the association of peptides intodimers. In another embodiment of the invention, portions ofimmunoglobulin gene sequences are used to drive the association ofnon-immunoglobulin peptides. These portions correspond to discretedomains of immunoglobulins. Immunoglobulins comprise variable andconstant regions, which in turn comprise discrete domains that showsimilarity in their three-dimensional conformations. These discretedomains correspond to immunoglobulin heavy chain constant region domainexons, immunoglobulin heavy chain variable region domain exons,immunoglobulin light chain varable region domain exons andimmunoglobulin light chain constant region domain exons inimmunoglobulin genes (Hood et al., in Immunology, The Benjamin/CummingsPublishing Company, Inc., Menlo Park, Calif.; Honjo et al., Cell 18:559-568, 1979; Takahashi et al., Cell 29: 671-679, 1982; and Honjo, Ann.Rev. Immun. 1:499-528, 1983)). Particularly preferred portions ofimmunoqlobulin heavy chains include Fab and Fab' fragments. (An Fabfragment is a portion of an immunoglobulin heavy chain that includes aheavy chain variable region domain and a heavy chain constant regiondomain. An Fab' fragment is a portion of an immunoglobulin heavy chainthat includes a heavy chain variable region domain, a heavy chainconstant region domain and a heavy chain hinge region domain.)

It is preferred to use an immunoglobulin light chain constant region inassociation with at least one immunoglobulin heavy chain constant regiondomain. In a another embodiment, an immunoglobulin light chain constantregion is associated with at least one immunoglobulin heavy chainconstant region domain joined to an immunoglobulin hinge region. In oneset of embodiments, an immunoglobulin light chain constant region joinedin frame with a peptide dimer or receptor analog and is associated withat least one heavy chain constant region. In a preferred set ofembodiments a variable region is joined upstream or and in properreading frame with at least one immunoglobulin heavy chain constantregion. In another set of embodiments, an immunoglobulin heavy chain isjoined in frame with a peptide dimer or receptor analog and isassociated with an immunoglobulin light chain constant region. In yetanother set of embodiments, a peptide dimer or receptor analog joined toat least one immunoglobulin heavy chain constant region joined to animmunoglobulin hinge region and is associated with an immunoglobulinlight chain constant region. In a preferred set of embodiments animmunoglobulin varable region is joined upstream of and in properreading frame with the immunoglobulin light chain constant region.

Immunoglobulin heavy chain constant region domains include C_(H) 1,C_(H) 2, C_(H) 3, and C_(H) 4 of any class of Immunoglobulin heavy chainincluding γ, α, ε, μ, and δ classes (Honjo, ibid., 1983) A particularlypreferred immunoglobulin heavy chain constant region domain is humanC_(H) 1. Immunoglobulin variable regions include V_(H), V.sub.κ, orV.sub.γ.

DNA sequences encoding immunoglobulins may be cloned from a variety ofgenomic or cDNA libraries known in the art. The techniques for isolatingsuch DNA sequences are conventional techniques and are well known tothose skilled in the art. Probes for isolating such DNA sequences may bebased on published DNA sequences (see, for example, Hieter et al., Cell22: 197-207, 1980). The choice of library and selection of probes forthe isolation of such DNA sequences is within the level of ordinaryskill in the art.

Host cells for use in practicing the present invention include mammalianand fungal cells. Fungal cells, including species of yeast (e.g.,Saccharomyces spp., Schizosaccharomyces spp.), or filamentous fungi(e.g., Aspergillus spp., Neurospora spp.) may be used as host cellswithin the present invention. Strains of the yeast Saccharomycescerevisiae are particularly preferred.

Suitable yeast vectors for use in the present invention include YRp7(Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEp13(Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs,Nature 275:104-108, 1978) and derivatives thereof. Such vectors willgenerally include a selectable marker, which may be one of any number ofgenes that exhibit a dominant phenotype for which a phenotypic assayexists to enable transformants to be selected. Preferred selectablemarkers are those that complement host cell auxotrophy, provideantibiotic resistance or enable a cell to utilize specific carbonsources, and include LEU2 (Broach et al. ibid.), URA3 (Botstein et al.,Gene 8: 17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasaki andBell, EP 171,142). Other suitable selectable markers include the CATgene, which confers chloramphenicol resistance on yeast cells.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080,1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982;Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Younget al., in Genetic Engineering of Microorganisms for Chemicals,Hollaender et al., (eds.), p. 355, Plenum, N.Y., 1982; Ammerer, Meth.Enzymol. 101: 192-201, 1983). In this regard, particularly preferredpromoters are the TPI1 promoter (Kawasaki, U.S. Pat. No. 4,599,311,1986) and the ADH2-4^(c) promoter (Russell et al., Nature 304: 652-654,1983 and Irani and Kilgore, described in pending, commonly assigned U.S.patent application Ser. Nos. 029,867, now abandoned, and 183,130, nowabandoned which are incorporated herein by reference). The expressionunits may also include a transcriptional terminator. A preferredtranscriptional terminator is the TPI1 terminator (Alber and Kawasaki,ibid.).

In addition to yeast, proteins of the present invention can be expressedin filamentous fungi, for example, strains of the fungi Aspergillus(McKnight and Upshall, described in pending, commonly assigned U.S.patent application Ser. Nos. 820,519, now abandoned, and 946,873, nowU.S. Pat. No. 4,035,349, corresponding to published European PatentApplication EP 272,277, which are incorporated herein by reference).Examples of useful promoters include those derived from Aspergillusnidulans glycolytic genes, such as the ADH3 promoter (McKnight et al.,EMBO J. 4: 2093-2099, 1985) and the promoter. An example of a suitableterminator is the ADH3 terminator (McKnight et al., ibid.). Theexpression units utilizing such components are cloned into vectors thatare capable of insertion into the chromosomal DNA of Aspergillus.

Techniques for transforming fungi are well known in the literature, andhave been described, for instance, by Beggs (ibid.), Hinnen et al.(Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978), Yelton et al., (Proc.Natl. Acad. Sci. USA 81: 1740-1747, 1984), and Russell (Nature 301:167-169, 1983). The genotype of the host cell will generally contain aqenetic defect that is complemented by the selectable marker present onthe expression vector. Choice of a particular host and selectable markeris well within the level of ordinary skill in the art.

In a preferred embodiment, a yeast host cell that contains a geneticdeficiency in a gene required for asparagine-linked glycosylation ofglycoproteins is used. Preferably, the yeast host cell contains agenetic deficiency in the MNN9 gene (described in pending, commonlyassigned U.S. patent application Ser. Nos. 116,095 and 189,547 which areincorporated by reference herein in their entirety). Most preferably,the yeast host cell contains a disruption of the MNN9 gene. Yeast hostcells having such defects may be prepared using standard techniques ofmutation and selection. Ballou et al. (J. Biol. Chem. 255: 5986-5991,1980) have described the isolation of mannoprotein biosynthesis mutantsthat are defective in genes which affect asparagine-linkedglycosylation. Briefly, mutagenized yeast cells were screened usingfluoresceinated antibodies directed against the outer mannose chainspresent on wild-type yeast. Mutant cells that did not bind antibody werefurther characterized and were found to be defective in the addition ofasparagine-linked oligosaccharide moieties. To optimize production ofthe heterologous proteins, it is preferred that the host strain carriesa mutation, such as the yeast pep4 mutation (Jones, Genetics 85: 23-33,1977), which results in reduced proteolytic activity.

In addition to fungal cells, cultured mammalian cells may be used ashost cells within the present invention. Preferred cell lines are rodentmyeloma cell lines, which include p3X63Ag8 (ATCC TIB 9), FO (ATCC CRL1646), NS-1 (ATCC TIB 18) and 210-RCY-Ag1 (Galfre et al., Nature 277:131, 1979). A particularly preferred rodent myeloma cell line isSP2/0-Ag14 (ATCC CRL 1581). In addition, a number of other cell linesmay be used within the present invention, including COS-1 (ATCC CRL1650), BHK, p363.Ag.8.653 (ATCC CRL 1580) Rat Hep I (ATCC CRL 1600), RatHep II (ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC CCL 75.1),Human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), Mouse liver (ATCCCC 29.1), 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72,1977) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci USA 77:4216-4220, 1980) A preferred BHK cell line is the tk⁻ ts13 BHK cell line(Waechter and Baserga, Proc. Natl. Acad. Sci USA 79: 1106-1110, 1982).

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Preferred viral promoters include the major latepromoter from adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-13199, 1982) and the SV40 promoter (Subramani et al., Mol. Cell.Biol. 1: 854-864, 1981). Preferred cellular promoters include the mousemetallothionein 1 promoter (Palmiter et al., Science 222: 809-814, 1983)and a mouse V.sub.κ promoter (Grant et al., Nuc. Acids Res. 15: 5496,1987). A particularly preferred promoter is a mouse VH promoter (Loh etal., ibid.). Such expression vectors may also contain a set of RNAsplice sites located downstream from the promoter and upstream from theDNA sequence encoding the peptide or protein of interest. Preferred RNAsplice sites may be obtained from adenovirus and/or immunoglobulingenes. Also contained in the expression vectors is a polyadenylationsignal located downstream of the coding sequence of interest.Polyadenylation signals include the early or late polyadenylationsignals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signalfrom the adenovirus 5 EIB region and the human growth hormone geneterminator (DeNoto et al., Nuc. Acids Res. 9: 3719-3730, 1981). Aparticularly preferred polyadenylation signal is the VH gene terminator(Loh et al., ibid.). The expression vectors may include a noncodingviral leader sequence, such as the adenovirus 2 tripartite leader,located between the promoter and the RNA splice sites. Preferred vectorsmay also include enhancer sequences, such as the SV40 enhancer and themouse enhancer (Gillies, Cell 33: 717-728, 1983). Expression vectors mayalso include sequences encoding the adenovirus VA RNAs.

Cloned DNA sequences may be introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603,1981; Graham and Van der Eb, Virology 52: 456, 1973.) Other techniquesfor introducing cloned DNA sequences into mammalian cells, such aselectroporation (Neumann et al., EMBO J. 1: 841-845, 1982), may also beused. In order to identify cells that have integrated the cloned DNA, aselectable marker is generally introduced into the cells along with thegene or cDNA of interest. Preferred selectable markers for use incultured mammalian cells include genes that confer resistance to drugs,such as neomycin, hygromycin, and methotrexate. The selectable markermay be an amplifiable selectable marker. A preferred amplifiableselectable marker is the DHFR gene. A particularly preferred amplifiablemarker is the DHFR^(r) cDNA (Simonsen and Levinson, Proc. Natl. Adac.Sci. USA 80: 2495-2499, 1983). Selectable markers are reviewed by Thilly(Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.) andthe choice of selectable markers is well within the level of ordinaryskill in the art.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as "carrier DNA" to the mixture which isintroduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time,typically 1-2 days, to begin expressing the DNA sequence(s) of interest.Drug selection is then applied to select for growth of cells that areexpressing the selectable marker in a stable fashion. For cells thathave been transfected with an amplifiable selectable marker the drugconcentration may be increased in a stepwise manner to select forincreased copy number of the cloned sequences, thereby increasingexpression levels.

Host cells containing DNA constructs of the present invention are grownin an appropriate growth medium. As used herein, the term "appropriategrowth medium" means a medium containing nutrients required for thegrowth of cells. Nutrients required for cell growth may include a carbonsource, a nitrogen source, essential amino acids, vitamins, minerals andgrowth factors. The growth medium will generally select for cellscontaining the DNA construct by, for example, drug selection ordeficiency in an essential nutrient which are complemented by theselectable marker on the DNA construct or co-transfected with the DNAconstruct. Yeast cells, for example, are preferably grown in achemically defined medium, comprising a non-amino acid nitrogen source,inorganic salts, vitamins and essential amino acid supplements. The pHof the medium is preferably maintained at a pH greater than 2 and lessthan 8, preferably at pH 6.5. Methods for maintaining a stable pHinclude buffering and constant pH control, preferably through theaddition of sodium hydroxide. Preferred buffering agents includesuccinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeastcells having a defect in a gene required for asparagine-linkedglycosylation are preferably grown in a medium containing an osmoticstabilizer. A preferred osmotic stabilizer is sorbitol supplemented intothe medium at a concentration between 0.1M and 1.5M., preferably at 0.5Mor 1.0M. Cultured mammalian cells are generally grown in commerciallyavailable serum-containing or serum-free media. Selection of a mediumappropriate for the particular cell line used is within the level ofordinary skill in the art.

The culture medium from appropriately grown transformed or transfectedhost cells is separated from the cell material, and the presence ofpeptide dimers or secreted receptor analogs is demonstrated. A preferredmethod of detecting receptor analogs, for example, is by the binding ofthe receptors or portions of receptors to a receptor-specific antibody.An anti-receptor antibody may be a monoclonal or polyclonal antibodyraised against the receptor in question, for example, an anti-PDGFreceptor monoclonal antibody may be used to assay for the presence ofPDGF receptor analogs. Another antibody, which may be used for detectingsubstance P-tagged peptides and proteins, is a commercially availablerat anti-substance P monoclonal antibody which may be utilized tovisualize peptides or proteins that are fused to the C-terminal aminoacids of substance P. Ligand binding assays may also be used to detectthe presence of receptor analogs. In the case of PDGF receptor analogs,it is preferable to use fetal foreskin fibroblasts, which express PDGFreceptors, to compete against the PDGF receptor analogs of the presentinvention for labeled PDGF and PDGF isoforms.

Assays for detection of secreted, biologically active peptide dimers andreceptor analogs may include Western transfer, protein blot or colonyfilter. A Western transfer filter may be prepared using the methoddescribed by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354,1979). Briefly, samples are electrophoresed in a sodium dodecylsulfatepolyacrylamide gel. The proteins in the gel are electrophoreticallytransferred to nitrocellulose paper. Protein blot filters may beprepared by filtering supernatant samples or concentrates throughnitrocellulose filters using, for example, a Minifold (Schleicher &Schuell). Colony filters may be prepared by growing colonies on anitrocellulose filter that has been laid across an appropriate growthmedium. In this method, a solid medium is preferred. The cells areallowed to grow on the filters for at least 12 hours. The cells areremoved from the filters by washing with an appropriate buffer that doesnot remove the proteins bound to the filters. A preferred buffercomprises 25 mM Tris-base, 19 mM glycine, pH 8.3, 20% methanol.

The peptide dimers and receptor analogs present on the Western transfer,protein blot or colony filters may be visualized by specific antibodybinding using methods known in the art. For example, Towbin et al.(ibid.) describe the visualization of proteins immobilized onnitrocellulose filters with a specific antibody followed by a labeledsecond antibody, directed against the first antibody. Kits and reagentsrequired for visualization are commercially available, for example, fromVector Laboratories, (Burlingame, Calif.), and Sigma Chemical Company(St. Louis, Mo.).

Secreted, biologically active peptide dimers and receptor analogs may beisolated from the medium of host cells grown under conditions that allowthe secretion of the receptor analogs and biologically active peptidedimers. The cell material is removed from the culture medium, and thebiologically active peptide dimers and receptor analogs are isolatedusing isolation techniques known in the art. Suitable isolationtechniques include precipitation and fractionation by a variety ofchromatographic methods, including gel filtration, ion exchangechromatography and immunoaffinity chromatography. A particularlypreferred purification method is immunoaffinity chromatography using anantibody directed against the receptor analog or peptide dimer. Theantibody is preferably immobilized or attached to a solid support orsubstrate. A particularly preferred substrate is CNBr-activatedSepharose (Pharmacia, Piscataway, N.J.). By this method, the medium iscombined with the antibody/substrate under conditions that will allowbinding to occur. The complex may be washed to remove unbound material,and the receptor analog or peptide dimer is released or eluted throughthe use of conditions unfavorable to complex formation. Particularlyuseful methods of elution include changes in pH, wherein the immobilizedantibody has a high affinity for the ligand at a first pH and a reducedaffinity at a second (higher or lower) pH; changes in concentration ofcertain chaotropic agents; or through the use of detergents.

The secreted PDGF receptor analogs of the present invention can be usedwithin a variety of assays for detecting the presence of native PDGF,PDGF isoforms or PDGF-like molecules. These assays will typicallyinvolve combining PDGF receptor analogs, which may be bound to a solidsubstrate such as polymeric microtiter plate wells, with a biologicalsample under conditions that permit the formation of receptor/ligandcomplexes. Detection may be achieved through the use of a label attachedto the receptor or through the use of a labeled antibody which isreactive with the receptor. Alternatively, the labeled antibody may bereactive with the ligand. A wide variety of labels may be utilized, suchas radionuclides, fluorophores, enzymes and luminescers. Complexes mayalso be detected visually, i.e., in immunoprecipitation assays, which donot require the use of a label.

Secreted PDGF receptor analogs of the present invention may also belabeled with a radioisotope or other imaging agent and used for in vivodiagnostic purposes. Preferred radioisotope imaging agents includeiodine-125 and technetium-99, with technetium-99 being particularlypreferred. Methods for producing protein-isotope conjugates are wellknown in the art, and are described by, for example, Eckelman et al.(U.S. Pat. No. 4,652,440), Parker et al. (WO 87/05030) and Wilber et al.(EP 203,764). Alternatively, the secreted receptor analogs may be boundto spin label enhancers and used for magnetic resonance (MR) imaging.Suitable spin label enhancers include stable, sterically hindered, freeradical compounds such as nitroxides. Methods for labeling ligands forMR imaging are disclosed by, for example, Coffman et al. (U.S. Pat. No.4,656,026). For administration, the labeled receptor analogs arecombined with a pharmaceutically acceptable carrier or diluent, such assterile saline or sterile water. Administration is preferably by bolusinjection, preferably intravenously. These imaging agents areparticularly useful in identifying the locations of atheroscleroticplaques, PDGF-producing tumors, and receptor-bound PDGF.

The secreted PDGF receptor analogs of the present invention may also beutilized within diagnostic kits. Briefly, the subject receptor analogsare preferably provided in a lyophilized form or immobilized onto thewalls of a suitable container, either alone or in conjunction withantibodies capable of binding to native PDGF or selected PDGF isoform(s)or specific ligands. The antibodies, which may be conjugated to a labelor unconjugated, are generally included in the kits with suitablebuffers, such as phosphate, stabilizers, inert proteins or the like.Generally, these materials are present in less than about 5% weightbased upon the amount of active receptor analog, and are usually presentin an amount of at least about 0.001% weight. It may also be desirableto include an inert excipient to dilute the active ingredients. Such anexcipient may be present from about 1% to 99% weight of the totalcomposition. In addition, the kits will typically include other standardreagents, instructions and, depending upon the nature of the labelinvolved, reactants that are required to produce a detectable product.Where an antibody capable of binding to the receptor or receptor/ligandcomplex is employed, this antibody will usually be provided in aseparate vial. The antibody is typically conjugated to a label andformulated in an analogous manner with the formulations brieflydescribed above. The diagnostic kits, including the containers, may beproduced and packaged using conventional kit manufacturing procedures.

As noted above, the secreted PDGF receptor analogs of the presentinvention may be utilized within methods for purifying PDGF from avariety of samples. Within a preferred method, the secreted PDGFreceptor analogs are immobilized or attached to a substrate or supportmaterial, such as polymeric tubes, beads, polysaccharide particulates,polysaccharide-containing materials, polyacrylamide or other waterinsoluble polymeric materials. Methods for immobilization are well knownin the art (Mosbach et al., U.S. Pat. No. 4,415,665; Clarke et al.,Meth. Enzymology 68: 436-442, 1979). A common method of immobilizationis CNBr activation. Activated substrates are commercially available froma number of suppliers, including Pharmacia (Piscataway, N.J.), PierceChemical Co. (Rockford, Ill. and Bio-Rad Laboratories (Richmond,Calif.). A preferred substrate is CNBr-activated Sepharose (Pharmacia,Piscataway, N.J.). Generally, the substrate/receptor analog complex willbe in the form of a column. The sample is then combined with theimmobilized receptor analog under conditions that allow binding tooccur. The substrate with immobilized receptor analog is firstequilibrated with a buffer solution of a composition in which thereceptor analog has been previously found to bind its ligand. Thesample, in the solution used for equilibration, is then applied to thesubstrate/receptor analog complex. Where the complex is in the form of acolumn, it is preferred that the sample be passed over the column two ormore times to permit full binding of ligand to receptor analog. Thecomplex is then washed with the same solution to elute unbound material.In addition, a second wash solution may be used to minimize nonspecificbinding. The bound material may then be released or eluted through theuse of conditions unfavorable to complex formation. Particularly usefulmethods include changes in pH, wherein the immobilized receptor has ahigh affinity for PDGF at a first pH and reduced affinity at a second(higher or lower) pH; changes in concentration of certain chaotropicagents; or through the use of detergents.

The secreted PDGF receptor analogs fused to dimerizing proteins of thepresent invention may be used in pharmaceutical compositions for topicalor intravenous application. The PDGF receptor analogs fused todimerizing proteins are used in combination with a physiologicallyacceptable carrier or diluent. Preferred carriers and diluents includesaline and sterile water. Pharmaceutical compositions may also containstabilizers and adjuvants. The resulting aqueous solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile aqueoussolution prior to administration.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

Enzymes, including restriction enzymes, DNA polymerase I (Klenowfragment), T4 DNA polymerase, T4 DNA ligase and T4 polynucleotidekinase, were obtained from New England Biolabs (Beverly, Mass.),Bethesda Research Laboratories (Gaithersburg, Md.) andBoerhinger-Mannheim Biochemicals (Indianapolis, Ind.) and were used asdirected by the manufacturer or as described in Maniatis et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., 1982).

EXAMPLE 1 Cloning of the PDGF Receptor cDNA

Complementary DNA (cDNA) libraries were prepared from poly(A) RNA fromdiploid human dermal fibroblast cells, prepared by explant from a normaladult, essentially as described by Hagen et al. (Proc. Natl. Acad. Sci.USA 83: 2412-2416, 1986). Briefly, the poly(A) RNA was primed witholigo(T) and cloned into kgt11 using Eco RI linkers. The random primedlibrary was screened for the presence of human PDGF receptor cDNA'susing three oligonucleotide probes complementary to sequences of themouse PDGF receptor (Yarden et al., Nature 323: 226-232, 1986).Approximately one million phage form the random primed human fibroblastcell library were screened using oligonucleotides ZC904, ZC905 and ZC906(Table 1). Eight positive clones were identified and plaque purified.Two clones, designated RP41 and RP51, were selected for further analysisby restriction enzyme mapping and DNA sequence analysis. RP51 was foundto contain 356 bp of 5'-noncoding sequence, the ATG translationinitiation codon and 738 bp of the amino terminal coding sequence. RP41was found to overlap clone RP51 and contained 2649 bp encoding aminoacids 43-925 of the receptor protein.

                                      TABLE 1                                     __________________________________________________________________________    Oligonucleotides Sequences                                                    __________________________________________________________________________    ZC582                                                                              5' AAT TCC CGG G 3'                                                      ZC583                                                                              5' GAT CCC CGG G 3'                                                      ZC871                                                                              5' CTC TCT TCC TCA GGT AAA TGA GTG CCA GGG CCG                                GCA AGC CCC CGC TCC 3'                                                   ZC872                                                                              5' CCG GGG AGC GGG GGC TTG CCG GCC CTG GCA CTC                                ATT TAC CTG AGG AAG AGA GAG CT 3'                                        ZC904                                                                              5' CAT GGG CAC GTA ATC TAT AGA TTC ATC CTT GCT                                CAT ATC CAT GTA 3'                                                       ZC905                                                                              5' TCT TGC CAG GGC ACC TGG GAC ATC TGT TCC CAC                                ATG ACC GG 3'                                                            ZC906                                                                              5' AAG CTG TCC TCT GCT TCA GCC AGA GGT CCT GGG                                CAG CC 3'                                                                ZC1380                                                                             5' CAT GGT GGA ATT CCT GCT GAT 3'                                        ZC1447                                                                             5' TG GTT GTG CAG AGC TGA GGA AGA GAT GGA 3'                             ZC1453                                                                             5' AAT TCA TTA TGT TGT TGC AAG CCT TCT TGT TCC                                TGC TAG CTG GTT TCG CTG TTA A 3'                                         ZC1454                                                                             5' GAT CTT AAC AGC GAA ACC AGC TAG CAG GAA CAA                                GAA GGC TTG CAA CAA CAT AAT G 3'                                         ZC1478                                                                             5' ATC GCG AGC ATG CAG ATC TGA 3'                                        ZC1479                                                                             5'AGC TTC AGA TCT GCA TGC TGC CGA T                                      ZC1776                                                                             5' AGC TGA GCG CAA ATG TTG TGT CGA GTG CCC ACC                                GTG CCC AGC TTA GAA TTC T 3'                                             ZC1777                                                                             5' CTA GAG AAT TGT AAG CTG GGC AAC GTG GGC ACT                                CGA CAC AAC ATT TGC GCT G 3'                                             ZC1846                                                                             5' GAT CGG CCA CTG TCG GTG CGC TGC ACG CTG CGC                                AAC GCT GTG GGC CAG GAC ACG CAG GAG GTC ATC                                   GTG GTG CCA CAC TCC TTG CCC TTT AAG CA 3'                                ZC1847                                                                             5' AGC TTG CTT AAA GGG CAA GGA GTG TGG CAC CAC                                GAT GAC CTC CTG CGT GTC CTG GCC CAC AGC GTT                                   GCG CAG CGT GCA GCG CAC CGA CAG TGG CC 3'                                ZC1886                                                                             5' CCA GTG CCA AGC TTG TCT AGA CTT ACC TTT AAA                                GGG CAA GGA G 3'                                                         AC1892                                                                             5' AGC TTG AGC GT 3'                                                     ZC1893                                                                             5' CTA GAC GCT CA 3'                                                     ZC1894                                                                             5' AGC TTC CAG TTC TTC GGC CTC ATG TCA GTT CTT                                CGG CCT CAT GTG AT 3'                                                    ZC1895                                                                             5' CTA GAT CAC ATG AGG CCG AAG AAC TGA CAT GAG                                GCC GAA GAA CTG GA 3'                                                    __________________________________________________________________________

The 3'-end of the cDNA was not isolated in the first cloning and wassubsequently isolated by screening 6×10⁵ phage of the oligo (dT)-primedcDNA library with a 630 bp Sst I-Eco RI fragment derived from the 3'-endof clone RP41. One isolate, designated OT91, was further analyzed byrestriction enzyme mapping and DNA sequencing. This clone was found tocomprise the 3'-end of the receptor coding region and 1986 bp of3'-untranslated sequence.

Clones RP51, RP41 and OT91 were ligated together to construct afull-length cDNA encoding the entire PDGF receptor. RP41 was digestedwith Acc I and Bam HI to isolate the 2.12 kb fragment. RP51 was digestedwith Eco RI and Acc I to isolate the 982 bp fragment. The 2.12 kb RP41fragment and the 982 bp RP51 fragment were joined in a three-partligation with pUC13, which had been linearized by digestion with Eco RIand Bam HI. The resultant plasmid Was designated 51/41. plasmid 51/41was digested with Eco RI and Bam HI to isolate the 3 kb fragmentcomprising the partial PDGF receptor cDNA. OT91 was digested with Bam HIand Xba I to isolate the 1.4 kb fragment containing the 3' portion ofthe PDGF receptor cDNA. The Eco RI-Bam HI 51/41 fragment, the Bam HI-XbaI OT91 fragment and the Eco RI-Xba I digested pUC13 were joined in athree-part ligation. The resultant plasmid was designated pR-RXI (FIG.2).

EXAMPLE 2 Construction of a SUC2 Signal Sequence-PDGF Receptor Fusion

To direct the PDGF receptor into the yeast secretory pathway, the PDGFreceptor cDNA was joined to a sequence encoding the Saccharomvcescerevisiae SUC2 signal sequence. Oligonucleotides ZC1453 and ZC1454(Table 1) were designed to form an adapter encoding the SUC2 secretorysignal flanked by a 5' Eco RI adhesive end and a 3, Bgl II adhesive end.ZC1453 and ZC1454 were annealed under conditions described by Maniatiset al. (ibid.). Plasmid pR-RX1 was digested with Bgl II and Sst II toisolate the 1.7 kb fragment comprising the PDGF receptor coding sequencefrom amino acids 28 to 596. Plasmid pR-RX1 was also cut with Sst II andHind III to isolate the 1.7 kb fragment comprising the coding sequencefrom amino acids 597 through the translation termination codon and 124bp of 3' untranslated DNA. The two 1.7 kb pR-RX1 fragments and theZC1453/ZC1454 adapter were joined with pUC19, which had been linearizedby digestion with Eco RI and Hind III. The resultant plasmid, comprisingthe SUC2 signal sequence fused in-frame with the PDGF receptor cDNA, wasdesignated pBTL10 (FIG. 2).

EXAMPLE 3 Construction of pCBS22

The BAR1 gene product, Barrier, is an exported protein that has beenshown to have three domains. When used in conjunction with a firstsignal sequence, the third domain of Barrier protein has been shown toaid in the secretion of proteins into the medium (MacKay et al., U.S.patent application Ser. No. 104,316).

The portion of the BAR1 gene encoding the third domain of Barrier wasjoined to a sequence encoding the C-terminal portion of substance P(subP; Munro and Pelham, EMBO J. 3: 3087-3093, 1984). The presence ofthe substance P amino acids on the terminus of the fusion proteinallowed the protein to be detected using commercially availableanti-substance P antibodies. Plasmid pZV9 (deposited as a transformantin E. coli strain RR1, ATCC accession no. 53283), comprising the entireBAR1 coding region and its associated flanking regions, was cut with SalI and Bam HI to isolate the 1.3 kb BAR1 fragment. This fragment wassubcloned into pUC13, which had been cut with Sal I and Bam HI, togenerate the plasmid designated pZV17. Plasmid pZV17 was digested withEco RI to remove the 3'-most 0.5 kb of the BAR1 coding region. Thevector-BAR1 fragment was religated to create the plasmid designatedpJH66 (FIG. 3). Plasmid pJH66 was linearized with Eco RI and blunt-endedwith DNA polymerase I (Klenow fragment). Kinased Bam HI linkers (5' CCGGAT CCG G 3') were added and excess linkers were removed by digestionwith Bam HI before religation. The resultant plasmid was designated pSW8(FIG. 3).

Plasmid pSW81, comprising the promoter, the BAR1 coding region fused tothe coding region of the C-terminal portion of substance P (Munro andPelham, EMBO J. 3: 3087-3093, 1984) and the TPI1 terminator, was derivedfrom pSW8. Plasmid pSW8 was cut with Sal I and Bam HI to isolate the 824bp fragment encoding amino acids 252 through 526 of BAR1. Plasmid pPM2,containing the synthetic oligonucleotide sequence encoding the dimerform of the C-terminal portion of substance P (subP) in M13mp8, wasobtained from Hugh Pelham (MRC Laboratory of Molecular Biology,Cambridge, England). Plasmid pPM2 was linearized by digestion with BamHI and Sal I and ligated with the 824 bp BAR1 fragment from pSW8. Theresultant plasmid, pSWI4, was digested with Sal I and Sma I to isolatethe 871 bp BAR1-substance P fragment. Plasmid pSW16, comprising afragment of BAR1 encoding amino acids 1 through 250, was cut with Xba Iand Sal I to isolate the 767 bp BAR1 fragment. This fragment was ligatedwith the 871 bp BAR1-substance P fragment in a three-part ligation withpUC18 cut with Xba I and Sma I. The resultant plasmid, designated pSW15,was digested with Xba I and Sma I to isolate the 1.64 kb BAR1-substanceP fragment. The ADH1 promoter was obtained from pRL029. Plasmid pRL029,comprising the ADH1 promoter and the BAR15' region encoding amino acids1 to 33 in pUC18, was digested with Sph I and Xba I to isolate the 0.42kb ADH1 promoter fragment. The TPI1 terminator (Alber and Kawasaki,ibid.) was provided as a linearized fragment containing the TPI1terminator and pUC18 with a Klenow-filled Xba I end and an Sph I end.This fragment was ligated with the 0.42 kb ADH1 promoter fragment andthe 1.64 kb BAR1-substance P fragment in a three-part ligation toproduce plasmid pSW22.

The ADH1 promoter and the coding region of from the translationinitiation ATG through the Eco RV site present in pSW22, were removed bydigestion with Hind III and Eco RV. The 3.9 kb vector fragment,comprising the 401 bp between the Eco RV and the Eco RI sites of theBAR1 gene fused to subP and the TPI1 terminator, was isolated by gelelectrophoresis. Oligonucleotide ZC1478 (Table 1) was kinased andannealed with oligonucleotide ZC1479 (Table 1) using conditionsdescribed by Maniatis et al. (ibid.). The annealed oligonucleotidesformed an adapter comprising a Hind III adhesive end and a polylinkerencoding Bgl II, Sph I, Nru I and Eco RV restriction sites. TheZC1479/ZC1478 adapter was ligated with the gel-purified pSW22 fragment.The resultant plasmid was designated pCBS22 (FIG. 3).

EXAMPLE 4 Construction of pBTL13

In order to enhance the secretion of the receptor and to facilitate theidentification of the secreted protein, a sequence encoding the thirddomain of BAR1 fused to the C-terminal amino acids of substance P wasfused in frame with the 5' 1240 bp of the PDGF receptor. Plasmid pBTL10(Example 2) was digested with Sph I and Sst I to isolate the 4 kbfragment comprising the SUC2 signal sequence, a portion of the PDGFreceptor cDNA and the pUC19 vector sequences. Plasmid pCBS22 wasdigested with Sph I and Sst I to isolate the 1.2 kb fragment comprisingthe BAR1-subP fusion and the TPI1 terminator. These two fragments wereligated, and the resultant plasmid was designated pBTL13 (FIG. 4).

EXAMPLE 5 Construction of an Expression Vector Encoding the Entire PDGFReceptor

The entire PDGF receptor was directed into the secretory pathway byfusing a SUC2 signal sequence to the 5' end of the PDGF receptor codingsequence. This fusion was placed behind the TPI1 promoter and insertedinto the vector YEp13 for expression in yeast.

The TPI1 promoter was obtained from plasmid pTPIC10 (Alber and Kawasaki,J. Mol. Appl. Genet. 1: 410-434, 1982), and plasmid pFATPOT (Kawasakiand Bell, EP 171,142; ATCC 20699). Plasmid pTPIC10 was cut at the uniqueKpn I site, the coding region was removed with Bal-31 exonuclease, andan Eco RI linker (sequence: GGA ATT CC) was added to the 3, end of thepromoter. Digestion with Bgl II and Eco RI yielded a TPI1 promoterfragment having Bgl II and Eco RI sticky ends. This fragment was thenjoined to plasmid YRp7, (Stinchcomb et al., Nature 282: 39-43, 1979)that had been cut with Bgl II and Eco RI (partial). The resultingplasmid, TE32, was cleaved with Eco RI (partial) and Bam HI to remove aportion of the tetracycline resistance gene. The linearized plasmid wasthen recircularized by the addition of an Eco RI-Bam HI linker toproduce plasmid TEA32. Plasmid TEA32 was digested with Bgl II and EcoRI, and the 900 bp partial TPI1 promoter fragment was gel-purified.Plasmid pIC19H (Marsh et al., Gene 32:481-486, 1984) was cut with Bgl IIand Eco RI and the vector fragment was gel purified. The TPI1 promoterfragment was then ligated to the linearized pIC19H and the mixture wasused to transform E. coli RR1. Plasmid DNA was prepared and screened forthe presence of a ˜900 bp Bgl II-Eco RI fragment. A correct plasmid wasselected and designated pICTPIP.

The TPI1 promoter was then subcloned to place convenient restrictionsites at its ends. Plasmid pIC7 (Marsh et al., ibid.) was digested withEco RI, the fragment ends were blunted with DNA polymerase I (Klenowfragment), and the linear DNA was recircularized using T4 DNA ligase.The resulting plasmid was used to transform E. coli RR1. Plasmid DNA wasprepared from the transformants and was screened for the loss of the EcoRI site. A plasmid having the correct restriction pattern was designatedpIC7RI*. Plasmid pIC7RI* was digested with Hind III and Nar I, and the2500 bp fragment was gel-purified. The partial TPI1 promoter fragment(ca. 900 bp) was removed from pICTPIP using Nar I and Sph I and wasgel-purified. The remainder of the TPI1 promoter was obtained fromplasmid pFATPOT by digesting the plasmid with Sph I and Hind III, and a1750 bp fragment, which included a portion of the TPI1 promoter fragmentfrom pICTPIP, and the fragment from pFATPOT were then combined in atriple ligation to produce pMVR1 (FIG. 2).

The TPI1 promoter was then joined to the SUC2-PDGF receptor fusion.Plasmid pBTL10 (Example 2) was digested with Eco RI and Hind III toisolate the 3.4 kb fragment comprising the SUC2 signal sequence and theentire PDGF receptor coding region. Plasmid pMVR1 was digested with BglII and Eco RI to isolate the 0.9 kb TPI1 promoter fragment. The TPI1promoter fragment and the fragment derived from pBTL10 were joined withYEp13, which had been linearized by digestion with Bam HI and Hind III,in a three-part ligation. The resultant plasmid was designated pBTL12(FIG. 2).

EXAMPLE 6 Construction of an Expression Vector Encoding the 5'Extracellular Portion of the PDGF Receptor

The extracellular portion of the PDGF receptor was directed into thesecretory pathway by fusing the coding sequence to the signal sequence.This fusion was placed in an expression vector behind the TPI1 promoter.Plasmid pBTL10 (Example 2) was digested with Eco RI and Sph I to isolatethe approximately 1.3 kb fragment comprising the SUC2 signal sequenceand the PDGF receptor extracellular domain coding sequence. PlasmidpMVR1 (Example 5) was digested with Bgl II and Eco RI to isolate the 0.9kb TPI1 promoter fragment. The TPI1 promoter fragment was joined withthe fragment derived from pBTL10 and YEp13, which had been linearized bydigestion with Bam HI and Sph I, in a three-part ligation. The resultantplasmid was designated pBTLll (FIG. 2).

EXAMPLE 7 Construction of Yeast Expression Vectors pBTL14 and pBTL15,and The Expression of PDGF Receptor-BAR1-subP Fusions

A. Construction of pBTL14

The SUC2-PDGF-R fusion was joined with the third domain of BAR1 toenhance the secretion of the receptor, and the expression unit wascloned into a derivative of YEp13 termed pJH50. YEp13 was modified todestroy the Sal I site near the LEU2 gene. This was achieved bypartially digesting YEp13 with Sal I followed by a complete digestionwith Xho I. The 2.0 kb Xho I-Sal I fragment comprising the LEU2 gene andthe 8.0 kb linear YEp13 vector fragment were isolated and ligatedtogether. The ligation mixture was transformed into E. coli strain RR1.DNA was prepared from the transformants and was analyzed by digestionwith Sal I and Xho I. A clone was isolated which showed a single Sal Isite and an inactive Xho I site indicating that the LEU2 fragment hadinserted in the opposite orientation relative to the parent plasmidYEp13. The plasmid was designated pJH50.

Referring to FIG. 4, plasmid pBTL12 (Example 5) was digested with Sal Iand Pst I to isolate the 2.15 kb fragment comprising 270 bp of YEp13vector sequence, the TPI1 promoter, the SUC2 signal sequence, and 927 bpof PDGF receptor cDNA. Plasmid pBTL13 (Example 4) was digested with PstI and Hind III to isolate the 1.48 kb fragment comprising 313 bp of PDGFreceptor cDNA, the BAR1-subP fusion and the TPI1 terminator. Thefragments derived from pBTL12 and pBTL13 were joined with pJH50, whichhad been linearized by digestion with Hind III and Sal I, in athree-part ligation. The resultant plasmid was designated pBTL14.

B. Construction of pBTL15

Referring to FIG. 5, a yeast expression vector was constructedcomprising the TPI1 promoter, the SUC2 signal sequence, 1.45 kb of PDGFreceptor cDNA sequence fused to the fusion and the TPI1 terminator.Plasmid pBTL12 (Example 5) was digested with Sal I and Fsp I to isolatethe 2.7 kb fragment comprising the TPI1 promoter, the SUC2 signalsequence, the PDGF-R coding sequence, and 270 bp of YEp13 vectorsequence. Plasmid pBTL13 (Example 4) was digested with Nru I and HindIII to isolate the 1.4 kb fragment comprising the BAR1-subP fusion, theTPI1 terminator and 209 bp of 3' PDGF receptor cDNA sequence. Thefragments derived from pBTL12 and pBTL13 were joined in a three-partligation with pJH50, which had been linearized by digestion with HindIII and Sal I. The resultant plasmid was designated pBTL15.

C. Expression of PDGF-R-subP fusions from pBTL14 and pBTL15

Yeast expression vectors pBTL14 and pBTL15 were transformed intoSaccharomvces cerevisiae strains ZY100 (MATa leu2-3,112 ade2-101 suc2-Δ9gal2 pep4::TPI1prom-CAT) and ZY400 (MATa leu2-3, 112 ade2-101 suc2-Δ9gal2 pep4::TPI1prom-CAT Δmnn9::URA3). Transformations were carried outusing the method essentially described by Beggs (ibid.). Transformantswere selected for their ability to grow on -LEUDS (Table 2).

Table 2 Media Recipes

-LeuThrTrp Amino Acid Mixture

4 g adenine

3 g L-arginine

5 g L-aspartic acid

2 g L-histidine free base

6 g L-isoleucine

4 g L-lysine-mono hydrochloride

2 g L-methionine

6 g L-phenylalanine

5 g L-serine

5 g L-tyrosine

4 g uracil

6 g L-valine

Mix all the ingredients and grind with a mortar and pestle until themixture is finely ground.

-LEUDS

20 g glucose

6.7 g Yeast Nitrogen Base without amino acids (DIFCO LaboratoriesDetroit, Mich.)

0.6 g -LeuThrTrp Amino Acid Mixture

182.2 g sorbitol

18 g Agar

Mix all the ingredients in distilled water. Add distilled water to afinal volume of 1 liter. Autoclave 15 minutes. After autoclaving add 150mg L-threonine and 40 mg L-tryptophan. Pour plates and allow tosolidify.

-LEUDS+sodium succinate, pH 6.5

20 g Yeast Nitrogen Base without amino acids

0.6 g -LeuTrpThr Amino Acid Mixture

182.2 g sorbitol

11.8 g succinic acid

Mix all ingredients in distilled water to a final volume of 1 liter.Adjust the pH of the solution to pH 6.5. Autoclave 15 minutes. Afterautoclaving add 150 mg L-threonine and 40 mg L-tryptophan.

Fermentation Medium

7 g/l yeast nitrogen base without amino acids or ammonium sulfate (DIFCOLaboratories)

0.6 g/l ammonium sulfate

0.5 M sorbitol

0.39 g/l adenine sulfate

0.01% polypropylene glycol

Mix all ingredients in distilled water. Autoclave 15 minutes. Add 80 ml50% glucose for each liter of medium.

Super Synthetic -LEUD, pH 6.5 (liquid or solid medium)

6.7 g Yeast Nitrogen Base without amino acids or ammonium sulfate(DIFCO)

6 g ammonium sulfate

160 g adenine

0.6 g -LeuThrTrp Amino Acid Mixture

20 g glucose

11.8 g succinic acid

Mix all ingredients and add distilled water to a final volume of 800 ml.Adjust the pH of the solution to pH 6.4. Autoclave 15 minutes. For solidmedium, add 18 g agar before autoclaving, autoclave and pour plates.

Super Synthetic -LEUDS, pH 6.4 (liquid or solid medium)

Use the same recipe as Super Synthetic -LEUD, pH 6.4, but add 182.2 gsorbitol before autoclaving.

YEPD

20 g glucose

20 g Bacto Peptone (DIFCO Laboratories)

10 g Bacto Yeast Extract (DIFCO Labloratories)

18 g agar

4 ml 1% adenine

8 ml 1% L-leucine

Mix all ingredients in distilled water, and bring to a final volume of 1liter. Autoclave 25 minutes and pour plates.

The transformants were assayed for binding to an anti-PDGF receptormonoclonal antibody (PR7212) or an anti-substance P antibody by proteinblot assay. ZY100[pBTL14] and ZY100[pBTL15] transformants were grownovernight at 30° C. in 5 ml Super Synthetic -LEUD, pH 6.4 (Table 2).ZY400[pBTL14] and ZY400[pBTL15] transformants were grown overnight at30° C. in 5 ml Super Synthetic -LEUDS, pH 6.4 (Table 2). The cultureswere pelleted by centrifugation and the supernatants were assayed forthe presence of secreted PDGF receptor anlogs by protein blot assayusing methods described in Example 13. Results of assays using PR7212are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Results of a protein blot probed with PR7212                                  Transformant:                                                                 ______________________________________                                        ZY100[pBTL14]           +                                                     ZY400[pBTL14]           ++                                                    ZY100[pBTL15]           +                                                     ZY499[pBTL15]           +                                                     ______________________________________                                    

EXAMPLE 8 Construction of a SUC2-PDGF-R Fusion Comprising the CompletePDGF-R Extracellular Domain

A. Construction of pBTL22

The PDGF-R coding sequence present in pBTLI10 was modified to delete thecoding region 3' to the extracellular PDGF-R domain. As shown in FIG. 6,plasmid pBTL10 was digested with Sph I and Bam HI and with Sph I and SstII to isolate the 4.77 kb fragment and the 466 bp fragment,respectively. The 466 bp fragment was then digested with Sau 3A toisolate the 0.17 kb fragment. The 0.17 kb fragment and the 4.77 kb werejoined by ligation. The resultant plasmid was designated pBTL21.

Plasmid pBTL21, containing a Bam HI site that was regenerated by theligation of the Bam HI and Sau 3A sites, was digested with Hind III andBam HI to isolate the 4.2 kb fragment. Synthetic oligonucleotides ZC1846(Table 1) and ZC1847 (Table 1) were designed to form an adapter encodingthe PDGF-R from the Sau 3A site after bp 1856 (FIG. 1) to the end of theextracellular domain at 1958 bp (FIG. 1), having a 5, Bam HI adhesiveend that destroys the Bam HI site and a 3, Hind III adhesive end.Oligonucleotides ZC1846 and ZC1847 were annealed under conditionsdescribed by Maniatis et. al. (ibid.). The 4.2 pBTL21 fragment and theZC1846/ZC1847 adapter were joined by ligation. The resultant plasmid,designated pBTL22, comprises the SUC2 signal sequence fused in properreading frame to the extracellular domain of PDGF-R in the vector pUC19(FIG. 6).

B. Construction of pBTL28

An in-frame translation stop codon was inserted immediately after thecoding region of the PDGF-R in pBTL22 using oligonucleotides ZC1892(Table 1) and ZC1893 (Table 1). These oligonucleotides were designed toform an adapter encoding a stop codon in-frame with the PDGF-R codingsequence from pBTL22 flanked by a 5, Hind III adhesive end and a 3, XbaI adhesive end. Plasmid pBTL22 was digested with Eco RI and Hind III toisolate the 1.6 kb SUC2-PDGF-R fragment. Plasmid pMVRI was digested withEco RI and Xba I to isolate the 3.68 kb fragment comprising the TPI1promoter, pIC7RI* vector sequences and the TPI1 terminator.Oligonucleotides ZC1892 and ZC1893 were annealed to form a Hind III-XbaI adapter. The 1.6 kb SUC2-PDGF-R fragment, the 3.86 kb pMVRI fragmentand the ZC1892/ZC1893 adapter were joined in a three-part ligation. Theresultant plasmid was designated pBTL27.

The expression unit present in pBTL27 was inserted into the yeastexpression vector pJH50 by first digesting pJH50 with Bam HI and Sal Ito isolate the 10.3 kb vector fragment. Plasmid pBTL27 was digested withBgl II and Eco RI and with Xho I and Eco RI to isolate the 0.9 kb TPI1promoter fragment and the 1.65 kb fragment, respectively. The 10.3 kbpJH50 vector fragment, the 0.9 kb TPI1 promoter fragment and 1.65 kbfragment were joined in a three-part ligation. The resultant plasmid wasdesignated pBTL28.

C. Construction of Plasmid pBTL30

The PDGF-R coding sequence present in plasmid pBTL22 was modified toencode the twelve C-terminal amino acids of substance P and an in-framestop codon. Plasmid pBTL22 was digested with Eco RI and Hind III toisolate the 1.6 kb SUC2-PDGF-R fragment. Plasmid pMVR1 was digested withEco RI and Xba I to isolate the 3.68 kb fragment comprising the TPI1promoter, pIC7RI* and the TPI1 terminator. Synthetic oligonucleotidesZC1894 (Table 1) and ZC1895 (Table 1) were annealed to form an adaptercontaining the codons for the twelve C-terminal amino acids of substanceP followed by an in-frame stop codon and flanked on the 5, end with aHind III adhesive end and on the 3' end with an Xba I adhesive end. TheZC1894/ZC1895 adapter, the 1.6 kb SUC2-PDGF-R fragment and the pMVRIfragment were joined in a three-part ligation. The resultant plasmid,designated pBTL29, was digested with Eco RI and Xho I to isolate the1.69 kb SUC2-PDGF-R-subP-TPI1 terminator fragment. Plasmid pBTL27 wasdigested with Bgl II and Eco RI to isolate the 0.9 kb TPI1 promoterfragment. Plasmid pJH50 was digested with Bam HI and Sal I to isolatethe 10.3 kb vector fragment. The 1.69 kb pBTL29 fragment, the 0.9 kbTPI1 promoter fragment and the 10.3 kb vector fragment were joined in athree-part ligation. The resulting plasmid was designated pBTL30.

EXAMPLE 9 Construction and Expression of a SUC2-PDGF-R-IgG HingeExpression Vector

An expression unit comprising the TPI1 promoter, the SUC2 signalsequence, the PDGF-R extracellular domain, an immunoglobulin hingeregion and the SUC2 terminator was constructed. Plasmid pBTL22 wasdigested with Eco RI and Hind III to isolate the 1.56 kb fragment.Plasmid pMVRI was digested with Eco RI and Xba I to isolate the 3.7 kbfragment, comprising the TPI1 promoter, pIC7RI* vector sequences and theTPI1 terminator. Oligonucleotides ZC1776 (Table 1) and ZC1777 (Table 1)were designed to form, when annealed, an adapter encoding animmunoglobulin hinge region with a 5, Hind III adhesive end and a 3, XbaI adhesive end. Oligonucleotides ZC1776 and ZC1777 were annealed underconditions described by Maniatis et al. (ibid.). The 1.56 kb pBTL22fragment, the 3.7 kb fragment and the ZC1776/ZC1777 adapter were joinedin a three-part ligation, resulting in plasmid pBTL24.

The expression unit of pBTL24, comprising the TPI1 promoter, SUC2 signalsequence, PDGF-R extracellular domain sequence, hinge region sequence,and TPI1 terminator, was inserted into pJH50. Plasmid pBTL24 wasdigested with Xho I and Hind III to isolate the 2.4 kb expression unit.Plasmid pJH50 was digested with Hind III and Sal I to isolate the 9.95kb fragment. The 2.4 kb pBTL24 fragment and 9.95 kb pJH50 vectorfragment were joined by ligation. The resultant plasmid was designatedpBTL25.

Plasmid pBTL25 was transformed into Saccharomyces cerevisiae strainZY400 using the method essentially described by Beggs (ibid.).Transformants were selected for their ability to grow on -LEUDS (Table2). The transformants were tested for their ability to bind theanti-PDGF-R monoclonal antibody PR7212 using the colony assay methoddescribed in Example 13. Plasmid pBTL25 transformants were patched ontonitrocellulose filters that had been wetted and supported by YEPD solidmedium. Antibody PR7212 was found to bind to the PDGF-R-IgG hinge fusionsecreted by ZY400[pBTL25] transformants.

EXAMPLE 10 Construction and Expression of a SUC2 signal sequence-PDGF-RExtracellular Domain-SUC2 Fusion

As shown in FIG. 6, an expression unit comprising the TPI1 promoter,SUC2 signal sequence, PDGF-R extracellular domain sequence, and SUC2coding sequence was constructed as follows. Plasmid pBTL22 was digestedwith Eco RI and Hind III to isolate the 1.6 kb SUC2-PDGF-R fragment.Plasmid pMVRl was digested with Bgl II and Eco RI to isolate the 0.9 kbTPI1 promoter fragment. The SUC2 coding region was obtained from pJH40.Plasmid pJH40 was constructed by inserting the 2.0 kb Hind III-Hind IIISUC2 fragment from pRB58 (Carlson et al., Cell 28:145-154, 1982) intothe Hind III site of pUC19 followed by the destruction of the Hind IIIsite 3' to the coding region. Plasmid pJH40 was digested with Hind IIIand Sal I to isolate the 2.0 kb SUC2 coding sequence. Plasmid pJH50 wasdigested with Sal I and Bam HI to isolate the 10.3 kb vector fragment.The 0.9 kb Bgl II-Eco RI TPI1 promoter fragment, the 1.6 kb Eco RI-HindIII SUC2-PDGF-R, the 2.0 kb Hind III-Sal I SUC2 fragment and the 10.3 kbBam HI-Sal I vector fragment were joined in a four-part ligation. Theresultant plasmid was designated pBTL26 (FIG. 6).

Plasmid pBTL26 was transformed into Saccharomyces cerevisae strain ZY400using the method essentially described by Beggs (ibid.). Transformantswere selected for their ability to grow on -LEUDS (Table 2). ZY400transformants (ZY400[pBTL26]) were assayed by protein blot (Example 13),colony blot (Example 13) and competition assay.

Protein blot assays were carried out on ZY400[pBTL26] and ZY400[pJH50](control) transformants that had been grown in flasks. Two hundred-fiftyμl of a 5 ml overnight cultures of ZY400[pBTL26] in -LEUDS+sodiumsuccinate, pH 6.5 (Table 2) were inoculated into 50 ml of -LEUDS+ sodiumsuccinate, pH 6.5. The cultures were incubated for 35 hours in anairbath shaker at 30° C. The culture supernatants were harvested bycentrifugation. The culture supernatants were assayed as described inExample 13 and were found to bind PR7212 antibody.

Colony assays were carried out on ZY400[pBTL26] transformants.ZY400[pBTL26] transformants were patched onto wetted nitrocellulosefilters that were supported on a YEPD plate. The colony assay carriedout as described in Example 13.A. showed that ZY400[pBTL26] antibodiesbound PR7212 antibodies.

Competition binding assays were carried out on ZY400[pBTL26] andZY400[pJH50] transformants. The transformants were grown in 2 liters offermentation medium (Table 2) in a New Brunswick Bioflo2 fermentor (NewBrunswick, Philadelphia, Pa.) with continuous pH control at pH 6.4. Thecultures were adjusted to pH 7.5 immediately prior to harvesting.Culture supernatants were concentrated in an Amicon concentrator(Amicon, San Francisco, Calif.) using an Amicon 10⁴ mw spiral filtercartridge. The concentrated supernatants were further concentrated usingAmicon Centriprep 10s. Fifteen ml of the concentrated supernatantsamples were added to the Centripreps, and the Centripreps were spun ina Beckman GRP centrifuge (Beckman Instruments Inc., Carlsbad, Calif.) atsetting 5 for a total of 60 minutes. The concentrates were removed fromthe Centriprep and were assayed in the competition assay.

The competition binding assay measured the amount of 125I-PDGF left tobind to fetal foreskin fibroblast cells after preincubation with theconcentrate containing the PDGF-R-SUC2 fusion protein. The concentratewas serially diluted in binding medium (Table 4). The dilutions weremixed with 0.5 ng of iodinated PDGF-AA, PDGF-BB or PDGF-AB, and themixtures were incubated for two hours at room temperature. Three hundredμg of unlabeled PDGF-BB was added to each sample mixture. The samplemixtures were added to 24-well plates containing confluent fetalforeskin fibroblast cells (AG1523, available from the Human GeneticMutant Cell Repository, Camden, N.J.). The cells were incubated with themixture for four hours at 4° C. The supernatants were aspirated from thewells, and the wells were rinsed three times with phosphate bufferedsaline that was held at 4+ C. (PBS; Sigma, St. Louis, Mo.). Five hundredμl of PBS+1% NP-40 was added to each well, and the plates were shaken ona platform shaker for five minutes. The cells were harvested and theamount of iodinated PDGF was determined. The results of the competitionbinding assay showed that the PDGF-R-SUC2 fusion protein was able tocompetetively bind all three isoforms of PDGF.

The PDGF-R produced from ZY400 [pBTL26] transformants was tested forcross reactivity to fibroblast growth factor (FGF) and transforminggrowth factor-β (TGF-β) using the competition assay essentiallydescribed above. Supernatant concentrates from ZY400 [pBTL26] and ZY400[JH50] (control) transformants were serially diluted in binding medium(Table 4). The dilutions were mixed with 7.9 ng of iodinated FGF or 14 L25 ng of iodinated TGF-β, and the mixtures were incubated for two hoursat room temperature. Fourteen μg of unlabeled FGF was added to eachmixture containing labeled FGF, and 7 μg of unlabeled TGF-β was added toeach mixture containing labeled TGF-β. The sample mixtures were added to24-well plates containing confuent human dermal fibroblast cells. (Humandermal fibroblast cells express both FGF receptors and TGFβ receptors.)The cells were incubated with the mixtures for four hours at 4° C. Fivehundred μl of PBS+1% NP-40 was added to each well, and the plates wereshaken on a platform shaker for five minutes. The cells were harvestedand the amount of iodinated FGF or TGF-β bound to the cells wasdetermined. The results of these assays showed that the PDGF-R-SUC2fusion protein did not cross react with FGF or TGF-β.

Table 4 Reagent Recipes

Binding Medium

500 ml Ham's F-12 medium

12 ml 1M HEPES, pH 7.4

5 ml 100×PSN (Penicillin/Streptomycin/Neomycin, Gibco)

1 gm rabbit serum albumin

Western Transfer Buffer

25 mM Tris, pH 8.3

19 mM glycine, pH 8.3

20% methanol

Western Buffer A

50 ml 1M Tris, pH 7.4

20 ml 0.25 mM EDTA, pH 7.0

5 ml 10% NP-40

37.5 ml 4M NaCl

2.5 g gelatin

The Tris, EDTA, NP-40 and NaCl were diluted to a final volume of oneliter with distilled water. The gelatin was added to 300 ml of thissolution and the solution was heated in a microwave until the gelatinwas in solution. The gelatin solution was added back to the remainder ofthe first solution and stirred at 4° C. until cool. The buffer wasstored at 4° C.

Western Buffer B

50 ml 1M Tris, pH 7.4

20 ml 0.25 M EDTA, pH 7.0

5 ml 10% NP-40

58.4 g NaCl

2.5 g gelatin

4 g N-lauroyl sarcosine

The Tris, EDTA, NP-40, and the NaCl were mixed and diluted to a finalvolume of one liter. The gelatin was added to 300 ml of this solutionand heated in a microwave until the gelatin was in solution. The gelatinsolution was added back to the original solution and the N-lauroylsarcosine was added. The final mixture was stirred at 4° C. until thesolids were completely dissolved. This buffer was stored at 4° C.

2× Loading Buffer

36 ml 0.5M Tris-HCl, pH 6.8

6 ml glycerol

16 ml 20% SDS

4 ml 0.5% Bromphenol Blue in 0.5 M Tris-HCl, pH 6.8

Mix all ingredients. Immediately before use, add 100 μlβ-mercaptoethanol to each 900 μl dye mix

EXAMPLE 11 Construction and Expression of PDGF Receptor Analogs From BHKcells

A. Construction of pBTL114 and pBTL115

The portions of the PDGF receptor extracellular domain present in pBTL14and pBTL15 were placed in a mammalian expression vector. Plasmids pBTL14and pBTL15 were digested with Eco RI to isolate the 1695 bp and 1905 bpSUC2 signal-PDGF-R-BAR1 fragments. The 1695 bp fragment and the 1905 bpfragment were each ligated to ZEM229R that had been linearized bydigestion with Eco RI.

The vector ZEM229R was constructed as shown in FIG. 10 from ZEM229.Plasmid ZEM229 is a pUC18-based expression vector containing a uniqueBam HI site for insertion of cloned DNA between the mousemetallothionein-1 promoter and SV40 transcription terminator and anexpression unit containing the SV40 early promoter, mouse dihydrofolatereductase gene, and SV40 transcription terminator. Zem229 was modifiedto delete the Eco RI sites flanking the Bam HI cloning site and toreplace the Bam HI site with a single Eco RI cloning site. The plasmidwas partially digested with Eco RI, treated with DNA polymerase I(Klenow fragment) and dNTPs, and religated. Digestion of the plasmidwith Bam HI followed by ligaion of the linearized plasmid with a BamHI-Eco I adapter resulted in a unique Eco RI cloning site. The resultantplasmid was designated Zem229R.

The ligation mixtures were transformed into E. coli strain RR1. PlasmidDNA was prepared and the plasmids were subjected to restriction enzymeanalysis. A plasmid having the 1695 bp pBTL14 fragment inserted intoZem229 R in the correct orientation was designated pBTL114 (FIG. 9). Aplasmid having the 1905 bp pBTL15 fragment inserted into Zem229R in thecorrect orientation was designated pBTL115 (FIG. 9).

B. Expression of secreted PDGF receptor analogs in tk⁻ ts13 BHK cells

Plasmids pBTL114 and pBTL115 were each transfected into tk⁻ ts13 cellsusing calcium phosphate preciptation (essentially as described by Grahamand van der Eb, J. Gen. Virol. 36: 59-72, 1977). The transfected cellswere grown in Dulbecco's modified Eagle's medium (DMEM) containing 10%fetal calf serum, 1× PSN antibiotic mix (Gibco 600-5640). 2.0 mML-glutamine. The cells were selected in 250 nM methotrexate (MTX) for 14days, and the resulting colonies were screened by the immunofilter assay(McCracken and Brown, Biotechnioues, 82-87, Mar./Apr. 1984). Plates wererinsed with PBS or No Serum medium (DMEM plus 1×PSN antibiotic mix).Teflon® mesh (Spectrum Medical Industries, Los Angeles, Calif.) was thenplaced over the cells. Nitrocellulose filters were wetted with PBS or NoSerum medium, as appropriate, and placed over the mesh. After six hoursincubation at 37° C., filters were removed and placed in Wester buffer A(Table 4) overnight at room temperature. The filters were developedusing the antibody PR7212 and the procedure described in Example 13. Thefilters showed that conditioned media from pBTL114-transfected andpBTL115-transfected BHK cells bound the PR7212 antibody indicating thepresence of biologically active secreted PDGF-R.

EXAMPLE 12 Expression of PDGF Receptor Analogs In Cultured Mouse MyelomaCells

A. Construction of pICμPRE8

The immunoglobulin μ heavy chain promoter and enhancer were sublconedinto pIC19H to provide a unique Hind III site 3' to the promoter.Plasmid pμ (Grosschedl and Baltimore, Cell 41: 885-897, 1985) wasdigested with Sal I and Eco RI to isolate the 3.1 kb fragment comprisingthe μ promoter. Plasmid pIC19H was linearized by digestion with Eco RIand Xho I. The μ promoter fragment and the linearized pIC19H vectorfragment were joined by ligation. The resultant plasmid, designatedpICμ3, was digested with Ava II to isolate the 700 bp promoter fragment.The 700 bp fragment was blunt-ended by treatment with DNA polymerase I(Klenow fragment) and deoxynucleotide triphosphates. Plasmid pIC19H waslinearized by digestion with Xho I, and the adhesive ends were filled inby treatment with DNA polymerase I (Klenow fragement) anddeoxynucleotide triphosphates. The blunt-ended Ava II fragment wasligated with the blunt-ended, linearized pIC19H, and the ligationmixture was transformed into E. coli JM83. Plasmid DNA was prepared fromthe transformants and was analyzed by restriction digest. A plasmid witha Bgl II site 5, to the promoter was designated pICμPR1(-). PlasmidpICμPR1(-) was digested with Hind III and Bgl II to isolate the 700 bppromoter fragment. Plasmid pIC19R was linearized by digestion with HindIII and Bam HI. The 700 bp promoter fragment was joined with thelinearized pIC19R by ligation. The resultant plasmid, designatedpICμPR7, comprised the μ promoter with an unique Sma I site 5, to thepromoter and a unique Hind III site 3, to the promoter.

The immunoglobulin heavy chain μ enhancer (Gillies et al., Cell 33:717-728, 1983) was inserted into the unique Sma I site to generateplasmid pICμPRE8. Plasmid pJ4 (obtained from F. Blattner, Univ.Wisconsin, Madison, Wis.), comprising the 1.5 kb Hind III-Eco RI μenhancer fragment in the vector pAT153 (Amersham, Arlington Heights,Ill.), was digested with Hind III and Eco RI to isolate the 1.5 kbenhancer fragment. The adhesive ends of the enhancer fragment werefilled in by treatment with T4 DNA polymerase and deoxynucleotidetriphosphates. The blunt-ended fragment and pICμPR7, which had beenlinearized by digestion with Sma I, were joined by ligation. Theligation mixture was transformed into E. coli RR1. Plasmid DNA wasprepared from the transformants, and the plasmids were analyzed byrestriction digests. A plasmid comprising the enhancer and the μpromoter was designated pICμPRE8 (FIG. 7).

B. Construction of pSDL114

The DNA sequence encoding the extracellular domain of the PDGF receptorwas joined with the DNA sequence encoding the human immunoglobulin lightchain constant region. The PDGF extracellular domain was obtained frommpBTL22, which comprised the Eco RI-Hind III fragment from pBTL22(Example 8.A.) cloned into Eco RI-Hind III cut M13mp18. Single strandedDNA was prepared from a mpBTL22 phage clone, and the DNA was subjectedto vitro mutagenesis using the oligonucleotide ZC1886 (Table 1) and themethod described by Kunkel (Proc. Natl. Acad. Sci. USA 82: 488-492,1985). A phage clone comprising the mutagenized PDGF-R with a donorsplice site (5' splice site) at the 3' end of the PDGF-R extracellulardomain was designated pBTLR-HX (FIG. 7).

The native PDGF-R signal sequence was obtained from pPR5. Plasmid pPR5,comprising 738 bp of 5' coding sequence with an Eco RI site immediately5' to the translation initiation codon, was constructed by in vitromutagenesis of the PDGF-R cDNA fragment from RP51 (Example 1).Replicative form DNA of RP51 was digested with Eco RI to isolate the1.09 kb PDGF-R fragment. The PDGF-R fragment was cloned into the Eco RIsite of M13mp18. Single stranded template DNA was prepared from a phageclone containing the PDGF-R fragment in the proper orientation. Thetemplate DNA was subjected to in vitro mutagenesis using oligonucleotideZC1380 (Table 1) and the method described by Zoller and Smith (Meth.Enzymol. 100: 468-500, 1983). The mutagenesis resulted in the placementof an Eco RI site immediately 5' to the translation initiation codon.Mutagenized phage clones were analyzed by dideoxy sequence analysis. Aphage clone containing the ZC1380 mutation was selected and replicativeform (Rf) DNA was prepared from the phage clone. The Rf DNA was digestedwith Eco RI and Acc I to isolate the 0.63 kb fragment. Plasmid pR-RXI(Example 1) was digested with Acc I and Eco RI to isolate the 3.7 kbfragment. The 0.63 kb fragment and the 3.7 kb fragment were joined byligation resulting in plasmid pPR5 (FIG. 7).

As shown in FIG. 7, the PDGF-R signal peptide and part of theextracellular domain were obtained from plasmid pPR5 as a 1.4 kb EcoRI-Sph I fragment. Replicative form DNA from phage clone pBTLR-HX wasdigested with Sph I and Hind III to isolate the approximately 0.25 kbPDGF-R fragment. Plasmid pUC19 was linearized by digestion with Eco RIand Hind III. The 1.4 kb Eco RI-Sph I PDGF-R fragment, the 0.25 kb SphI-Hind III fragment from pBTLR-HX and the Eco RI-Hind III cut pUC19 werejoined in a three-part ligation. The resultant plasmid, pSDL110, wasdigested with Eco RI and Hind III to isolate the 1.65 kb PDGF-Rfragment.

Plasmid pICHuC_(k) 3.9.11 was used as the source of the humanimmunoglobulin light chain gene (FIG. 7). The human immunoglobulin lightchain gene was isolated from a human genomic library using anoligonucleotide probe (5' TGT GAC ACT CTC CTG GGA GTT A 3'), which wasbased on a published human kappa C gene sequence (Hieter et al., Cell22: 197-207, 1980). The human light chain (kappa) constant region wassubcloned as a 1.1 kb Sph I-Hinf I genomic fragment of the human kappagene, which has been treated with DNA polymerase DNA I (Klenow Fragment)to fill in the Hinf I adhesive end, into Sph I-Hinc II cut pUC19. The1.1 kb human kappa constant region was susbsequently isolated as a 1.1kb Sph I-Bam HI fragment that was subcloned into Sph I-Bgl II cut pIC19R(Marsh et al., ibid.). The resultant plasmid was designated pICHuC_(k)3.9.11. Plasmid pICHuC_(k) 3.9.11 was digested with Hind III and Eco RIto isolate the 1.1 kb kappa constant region gene. Plasmid pIC19H waslinearized by digestion with Eco RI. The 1.65 kb PDGF-R fragment, the1.1 kb human kappa constant region fragment and the linearized pIC19Hwere joined in a three part ligation. The resultant plasmid, pSDL112,was digested with Bam HI and Cla I to isolate the 2.75 kb fragment.Plasmid pμPRE8 was linearized with Bgl II and Cla I. The 2.75 kbfragment and the linearized pμPRE8 were joined by ligation. Theresultant plasmid was designated pSDL114 (FIG. 7).

Plasmid pSDL114 was linearized by digestion with Cla I and wascotransfected with Pvu I-digested p416 into SP2/0-Ag14 (ATCC CRL 1581)by electroporation using the method essentially described by Neumann etal. (EMBO J. 1: 841-845, 1982). (Plasmid p416 comprises the Adenovirus 5ori, SV40 enhancer, Adenovirus 2 major late promoter, Adenovirus 2tripartite leader, 5' and 3' splice sites, the DHFR^(r) cDNA, the SV40polyadenylation signal and pML-1 (Lusky and Botchan, Nature 293: 79-81,981) vector sequences.) Transfectants were selected in growth mediumcontaining methotrexate.

Media from drug resistant clones were tested for the presence ofsecreted PDGF receptor analogs by enzyme-linked immunosorbant assay(ELISA). Ninety-six well assay plates were prepared by incubating 100 μlof 1 μg/ml polyclonal goat anti-human kappa chain (Cappel Laboratories)diluted in phosphate buffered saline (PBS; Sigma) overnight at 4° C.Excess antibody was removed by one wash with 0.5% Tween 20 in PBS. Onehundred μl of spent media was added to each well, and the well wereincubated for one hour at 4° C. Unbound proteins were removed by onewash with 0.5% Tween 20 in PBS. One hundred μl of peroxidase-conjugatedgoat anti human kappa antibody (diluted 1:1000 in a solution containing5% chicken serum (Gibco)+0.5% Tween 20 in PBS) was added to each welland the wells were incubated for one hour at 4° C. One hundred μl ofchromophore (100 μl ABTS (2,2'-Azinobis(3-ethylbenz-thiazoline sulfonicacid diammonium salt; Sigma)+1 μl 30% H202+12.5 ml citrate/phosphatebuffer (9.04 g/l citric acid, 10.16 g/l Na₂ HPO₄)) was added to eachwell, and the wells were incubated to 30 minutes at room temperature.The samples were measured at 405 nm. The results of the assay showedthat the PDFG-R analog secreted by the transfectants contained animmunoglobulin light chain.

Spent media from drug resistant clones was also tested for the presenceof secreted PDGF receptor analogs by immunoprecipitaiton. Approximatelyone million drug resistant transfectants were grown in DMEM mediumlacking cysteine+2% calf serum for 18 hours at 37° C. in the presence of50 μCI ³⁵ S-cysteine. Media was harvested from the labeled cells and 250μl of the spent media was assayed for binding to the anti-PDGF receptorantibody PR7212. PR7212 diluted in PBS was added to the media to a finalconcentration of 2.5 μg per 250 μl spent media. Five μl of rabbitanti-mouse Ig diluted in PBS was added to the PR7212/media mixtures. Theimmunocomplexes were precipitated by the addition of 50 μl 110% fixedStaph A (weight/volume in PBS). The immunocomplexes were analyzed on 8%SDS-polyacrylamide gels followed by autoradiography overnight at -70° C.The results of the immunoprecipitation showed that the PDGF receptoranlaog secreted by the transfectants was bound by the anti-PDGF receptorantibody. The combined results of the ELISA and immunoprecipitationassays showed that the PDGF receptor analog secreted by thetransfectants contained both the PDGF receptor ligand-binding domain andthe human light chain constant region.

C. Cotransfection of pSDL114 with an immunoglobulin heavy chain

Plasmid pSDL114 was cotransfected with pφ5V_(H) huC.sub.γ 71M-neo, whichencodes a neomycin resistance gene expression unit and a completemouse/human chimeric immunoglobulin heavy chain gene expression unit(10).

Plasmid pφ5V_(H) huC.sub.γ 1M-neo was constructed as follows. The mouseimmunoglobulin heavy chain gene was isolated from a lambda genomic DNAlibrary constructed from the murine hybridoma cell line NR-ML-05(Serafini et al., Eur. J. Nucl. Med. 14: 232, 1988) using anoligonucleotide probe designed to span the VH/D/JH junction (5' GCA TAGTAG TTA CCA TAT CCT CTT GCA CAG 3'). The human immunoglobulin gamma-1 Cgene was isolated from a human genomic library using a cloned humangamma-4 constant region gene (Ellison et al., DNA 11-18, 1981). Themouse immunoglobulin variable region was isolated as a 5.3 kb Sst I-HindIII fragment from the original phage clone and the human gamma-1 C genewas obtained from the original phage clone as a 6.0 kb Hind III-Xho Ifragment. The chimeric gamma-1 C gene was created by joining the V_(H)and C_(H) fragments via the common Hind III site and incorporating themwith the E. coli neomycin resistance gene expression unit into pIC19H toyield pφ5V_(H) huC.sub.γ 1M-neo.

Plasmid pSDL114 was linearized by digestion with ClaI and wasco-transfected into SP2/0-Ag14 cells with Asp718 linearized pφ5V_(H)huC.sub.γ 1M-neo. The transfectants were selected in growth mediumcontaining methotrexate and neomycin. Media from drug-resistant cloneswere tested for their ability to bind PDGF in a competition bindingassay.

The competition binding assay measured the amount of ¹²⁵ I-PDGF left tobind to human dermal fibroblast cells after preincubation with the spentmedia from pSDL114-pφ5V_(H) huC.sub.γ 1M-neo transfected cells. Themedia were serially diluted in binding medium (Table 4). The dilutionswere mixed with 0.5 ng of iodinated PDGF-BB or iodinated PDGF-AA and themixtures were incubated for two hours at room temperature. Three hundredμg of unlabeled PDGF-BB or unlabeled PDGF-AA was added to one tube fromeach series. The sample mixtures were added to 24 well plates containingconfluent human dermal fibroblast cells. The cells were incubated withthe mixture for four hours at 4° C. The supernatants were aspirated fromthe wells, and the wells were rinsed three times with phosphate bufferedsaline that was held a 4° C. (PBS; Sigma, St. Louis, Mo.). Five hundredμl of PBS +1% NP-40 was added to each well, and the plates were shakenon a platform shaker for five minutes. The cells were harvested and theamount of iodinated PDGF was determined. The results of the competitionbinding assay showed that the protein produced from pSDL114-pφ5V_(H)huC.sub.γ 1M-neo transfected cells was able to competetively bindPDGF-BB but did not bind PDGF-AA.

The PDGF receptor analog produced from a pSDL114/pφ5V_(H) huC.sub.γ1M-neo transfectant was assayed to determine if the receptor analog wasable to bind PDGF-BB to saturation. Eight and one half milliliters ofspent media containing the PDGF-R analogs from a pSDL114/pφ5V_(H)huC.sub.γ 1M-neo transfectant was added to 425 μl of SepharoseCl-4B-Protein A beads (Sigma), and the mixture was incubated for 10minutes at 4° C. The beads were pelleted by centrifugation and washedwith binding medium (Table 4). Following the wash the beads wereresuspended in 8.5 ml of binding media, and 0.25 ml aliquots weredispensed to 1.5 ml tubes. Binding reactions were prepared by addingiodinated PDGF-BB diluted in DMEM+10% fetal calf serum to the identicalaliquots of receptor-bound beads to final PDGF-BB concentrations ofbetween 4.12 pM and 264 pM. Nonspecific binding was determined by addinga 100 fold excess of unlabeled BB to an identical set of bindingreactions. Mixtures were incubated overnight at 4° C.

The beads were pelleted by centrifugation, and unbound receptor wasremoved with three washes in PBS. The beads were resuspended in 100 μlof PBS and were counted. Results of the assay showed that the PDGF-Ranalog was able to bind PDGF-BB with high affinity.

D. Construction of pSDL113.

As shown in FIG. 8, the DNA sequence encoding the extracellular domainof the PDGF receptor was joined with the DNA sequence encoding a humanimmunoglobulin heavy chain constant region joined to a hinge sequence.Plasmid pSDL110 was digested with Eco RI and Hind III to isolate the1.65 kb PDGF-R fragment. Plasmid pICHu₆₅ -1M was used as the source ofthe heavy chain constant region and hinge region. Plasmid pICHu.sub.γ-1M comprises the approximately 6 kb Hind III-Xho I fragment of a humangamma-1 C gene subcloned into pUC19 that had been linearized bydigestion with Hind III and SaI I. Plasmid pICHu.sub.γ -1M was digestedwith Hind III and Eco RI to isolate the 6 kb fragment encoding the humanheavy chain constant region. Plasmid pIC19H was linearized by digestionwith Eco RI. The 1.65 kb PDGF-R fragment, the 6 kb heavy chain constantregion fragment and the linearized pIC19H were joined in a three partligation. The resultant plasmid, pSDLIII, was digested with Bam HI toisolate the 7.7 kb fragment. Plasmid pμPRE8 was linearized with Bgl IIand was treated with calf intestinal phosphatase to preventself-ligation. The 7.7 kb fragment and the linearized pμPRE8 were joinedby ligation. A plasmid containing the insert in the proper orientationwas designated pSDL113 (FIG. 8).

Plasmid pSDL113 is linearized by digestion with Cla I and wascotransfected with Pvu I-digested p416 into SP2/0-Ag14 byelectroporation. Transfectants are selected in growth medium containingmethotrexate.

Media from drug resistant clones are tested for the presence of secretedPDGF-R analogs by immunoprecipitation using the method described inExample 12.B. Competition binding assays (Example 12.C.) are carried outon media samples to determine ligand binding activity.

E. Cotransfection of pSDL113 with an Immunoglobulin Light Chain Gene

Plasmid pSDL113 is linearized by digestion with Cla I and iscotransfected with pICφ5V.sub.κ HuC.sub.κ -Neo, which encodes a neomycinresistance gene and a mouse/human chimeric immunoglobulin light chaingene. The mouse immunoglobulin light chain gene was isolated from alambda genomic DNA library constructed from the murine hybridoma cellline NR-ML-05 (Woodhouse et al., ibid.) using an oligonucleotide probedesigned to span the V.sub.κ /J.sub.κ junction (5' ACC GAA CGT GAG AGGAGT GCT ATA A 3'). The human immunoglobulin light chain constant regiongene was isolated as described in Example 12.B. The mouse NR-ML-05immunoglobulin light chain variable region gene was subcloned from theoriginal mouse genomic phage clone into pIC19R as a 3 kb Xba I-Hinc IIfragment. The human kappa C gene was subcloned from the original humangenomic phage clone into pUC19 as a 2.0 kb Hind III-Eco RI fragment. Thechimeric kappa gene was created by joining the NR-ML-05 light chainvariable region gene and human light chain constant region gene via thecommon Sph I site and incoporating them with the E. coli neomycinresistance gene into pIC19H to yield pICφ5V.sub.κ HuC.sub.κ -Neo (FIG.9).

The linearized pSDL113 and pICφ5V.sub.κ HuC.sub.κ -Neo are transfectedinto SP2/0-Ag14 cells by electroporation. The transfectants are selectedin growth medium containing methotrexate and neomycin. Media fromdrug-resistant clones are tested for their ability to bind PDGF in acompetition binding assay.

F. Cotransfection of pSDL113 and pSDL114

A clone of SP2/0-Ag14 stably transfected with pSDL114 and p416 wasco-transfected with Cla I-digested pSDL113 and Bam HI-digested pIC-neoby electroporation. (Plasmid pIC-neo comprises the SV40 promoteroperatively linked to the E. coli neomycin resistance gene and pIC19Hvector sequences.) Transfected cells were selected in growth mediumcontaining methotrexate and G418. Media from drug-resistance clones weretested for their ability to bind PDGF in a competition binding assay asdescribed in Example 12.C. The results of the assay showed that thetransfectants secreted a PDGF receptor analog capable of competitivelybinding PDGF-BB.

G. Cotransfection of pSDL114 with Fab

A clone of SP2/0-AG14 stably transfected with pSDL114 and p416 wastransfected with the Fab region of the human gamma-4 gene (γ₄) inplasmid pφ5V_(H) Fab-neo. Plasmid pφ5V_(H) Fab-neo was constructed asfollows.

Plasmid p24BRH (Ellison et al., DNA 1:11, 1988) was digested with Xma Iand Eco RI to isolate the 0.2 kb fragment comprising the immunoglobulin3' untranslated region. Synthetic oligonucleotides ZC871 (Table 1) andZC872 (Table 1) were kinased and annealed using essentially the methodsdescribed by Maniatis et al. (ibid.). The annealed oligonucleotidesZC871/ZC872 formed an Sst I-Xma I adapter. The ZC871/ZC872 adapter, the0.2 kb p24BRH fragment and Sst I-Eco RI linearized pUC19 were joined ina three-part ligation to form plasmid pγ₄ 3+. Plasmid pγ₄ 3' waslinearized by digestion with Bam HI and Hind III. Plasmid p24BRH was cutwith Hind III and Bgl II to isolate the 0.85 kb fragment comprising theC_(H) 1 region. The pγ₄ 3' fragment and the Hind III-Bgl II p24BRHfragment were joined by ligation to form plasmid p24Fab. Plasmid pγ₄ Fabwas digested with Hind III and Eco RI to isolate the 1.2 kb fragmentcomprising γ₄ Fab. Plasmid pICneo, comprising the SV40 promoteroperatively linked to the neomycin resistance gene and pIC19H vectorsequences, was linearized by digestion with Sst I and Eco RI. Plasmidpφ5V_(H), comprising the mouse immunoglobulin heavy chain gene variableregion and pUC18 vector sequences, was digested with Sst I and Hind IIIto isolate the 5.3 kb V_(H) fragment. The linearized pICneo was joinedwith the 5.3 kb Sst I-Hind III fragment and the 1.2 kb Hind III-Eco RIfragment in a three-part ligation. The resultant plasmid was designatedpφ5V_(H) Fab-neo (FIG. 10).

A pSDL114/p416-transfected SP2/0-AG14 clone was transfected with ScaI-linearized pφ5V_(H) Fab-neo. Transfected cells were selected in growthmedium containing methotrexate and G418. Media from drug-resistantclones were tested for their ability to bind PDGF in a competitionbinding assay as described in Example 12.C. The results of the assayshowed that the PDGF receptor analog secreted from the transfectants wascapable of competitively binding PDGF-BB.

H. Cotransfection of pSDL114 with Fab'

A stably transfected SP2/0-AG14 isolate containing pSDL114 and p416 wastransfected with plasmid pWKI, which contained the Fab' portion of animmunoglobulin heavy chain gene. Plasmid pWKI was constructed asfollows.

The immunoglobulin gamma-1 Fab' sequence, comprising the C_(H) 1 andhinge regions sequences, was derived from the gamma-1 gene clonedescribed in Example 12.C. The gamma-1 gene clone was digested with HindIII and Eco RI to isolate the 3.0 kb fragment, which was subcloned intoHind III-Eco RI linearized M13mp19. Single-stranded template DNA fromthe resultant phage was subjected to site-directed mutagenesis usingoligonucleotide ZC1447 (Table 1) and essentially the method of Zollerand Smith (ibid.). A phage clone was identified having a ZC1447 induceddeletion resulting in the fusion of the hinge region to a DNA sequenceencoding the amino acidsAla-Leu-His-Asn-His-Tyr-Thr-Glu-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gly-Lysfollowed in-frame by a stop codon. Replicative form DNA from a positivephage clone was digested with Hind III and Eco RI to isolate the 1.9 kbfragment comprising the C_(H) I and hinge regions. Plasmid pφ5V_(H) wasdigested with Sst I and Hind III to isolate the 5.3 kb fragmentcomprising the mouse immunoglobulin heavy chain gene variable region.Plasmid pICneo was linearized by digestion with Sst I and Eco RI. Thelinearized pICneo was joined with the 5.3 kb Hind III-Sst I fragment andthe 1.9 kb Hind III-Eco RI fragment in a three-part ligation. Theresultant plasmid was designated pWKI (FIG. 10).

An SP2/0-AG14 clone stably transfected with pSDL114 and p416 wastransfected with Asp 718-linearized pWKI. Transfected cells wereselected by growth in medium containing methotrexate and G418. Mediasamples from transfected cells were assayed using the competition assaydescribed in Example 12.C. Results from the assays showed that thetransfected cells produced a PDGF receptor analog capable ofcompetetively binding PDGF-BB.

EXAMPLE 13 Assay Methods

A. Preparation of Nitrocellulose Filters for Colony Assay

Colonies of transformants were tested for secretion of PDGF receptoranalogs by first growing the cells on nitrocellulose filters that hadbeen laid on top of solid growth medium. Nitrocellulose filters(Schleicher & Schuell, Keene, N.H.) were placed on top of solid growthmedium and were allowed to be completely wetted. Test colonies werepatched onto the wetted filters and were grown at 30° C. forapproximately 40 hours. The filters were then removed from the solidmedium, and the cells were removed by four successive rinses withWestern Transfer Buffer (Table 4). The nitrocellulose filters weresoaked in Western Buffer A (Table 4) for one hour at room temperature ona shaking platform with two changes of buffer. Secreted PDGF-R analogswere visualized on the filters described below.

B. Preparation of Protein Blot Filters

A nitrocellulose filter was soaked in Western Buffer A (Table 4) withoutthe gelatin and placed in a Minifold (Schleicher & Schuell, Keene,N.H.). Five ml of culture supernatant was added without dilution to theMinifold wells, and the liquid was allowed to pass through thenitrocellulose filter by gravity. The nitrocellulose filter was removedfrom the minifold and was soaked in Western Buffer A (Table 3) for onehour on a shaking platform at room temperature. The buffer was changedthree times during the hour incubation.

C. Preparation of Western Blot Filters

The transformants were analyzed by Western blot, essentially asdescribed by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354,1979) and Gordon et al. (U.S. Pat. No. 4,452,901). Culture supernatantsfrom appropriately grown transformants were diluted with three volumesof 95% ethanol. The ethanol mixtures were incubated overnight at -70° C.The precipitates were spun out of solution by centrifugation in an SS-24rotor at 18,000 rpm for 20 minutes. The supernatants were discarded andthe precipitate pellets were resuspended in 200 μl of dH₂ O. Two hundredμl of 2× loading buffer (Table 4) was added to each sample, and thesamples were incubated in a boiling water bath for 5 minutes.

The samples were electrophoresed in a 15% sodium dodecylsulfatepolyacrylamide gel under non-reducing conditions. The proteins wereelectrophoretically transferred to nitrocellulose paper using conditionsdescribed by Towbin et al. (ibid.). The nitrocellulose filters were thenincubated in Western Buffer A (Table 4) for 75 minutes at roomtemperature on a platform rocker.

D. Processing the Filters for Visualization with Antibody

Filters prepared as described above were screened for proteins capableof binding a PDGF receptor specific monoclonal antibody, designatedPR7212. The filters were removed from the Western Buffer A (Table 4) andplaced in sealed plastic bags containing a 10 ml solution comprising 10μg/ml PR7212 monoclonal antibody diluted in Western Buffer A. Thefilters were incubated on a rocking platform overnight at 4° C. or forone hour at room temperature. Excess antibody was removed with three15-minute washes with Western Buffer A on a shaking platform at roomtemperature.

Ten μl biotin-conjugated horse anti-mouse antibody (Vector Laboratories,Burlingame, Calif.) in 20 ml Western Buffer A was added to the filters.The filters were re-incubated for one hour at room temperature on aplatform shaker, and unbound conjugated antibody was removed with threefifteen-minute washes with Western Buffer A.

The filters were pre-incubated for one hour at room temperature with asolution comprising 50 μl Vectastain Reagent A (Vector Laboratories) in10 ml of Western Buffer A that had been allowed to incubate at roomtemperature for 30 minutes before use. The filters were washed with onequick wash with distilled water followed by three 15-minute washes withWestern Buffer B (Table 4) at room temperature. The Western Buffer Bwashes were followed by one wash with distilled water.

During the preceding wash step, the substrate reagent was prepared.Sixty mg of horseradish peroxidase reagent (Bio-Rad, Richmond, Calif.)was dissolved in 20 ml HPLC grade methanol. Ninety ml of distilled waterwas added to the dissolved peroxidase followed by 2.5 ml 2M Tris, pH 7.4and 3.8 ml 4M NaCl. 100 μl of 30% H₂ O was added just before use. Thewashed filters were incubated with 75 ml of substrate and incubated atroom temperature for 10 minutes with vigorous shaking. After the 10minute incubation, the buffer was changed, and the filters wereincubated for an additional 10 minutes. The filters were then washed indistilled water for one hour at room temperature. Positives were scoredas those samples which exhibited coloration.

E. Processing the Filters For Visualization with an Anti-Substance PAntibody

Filters prepared as described above were probed with an anti-substance Pantibody. The filters were removed from the Western Buffer A and rinsedwith Western transfer buffer, followed by a 5-minute wash in phosphatebuffered saline (PBS, Sigma, St. Louis, Mo.). The filters were incubatedwith a 10 ml solution containing 0.5M 1-ethyl-3-3-dimethylamino propylcarbodiimide (Sigma) in 1.0M NH₄ Cl for 40 minutes at room temperature.After incubation, the filters were washed three times, for 5 minutes perwash, in PBS. The filters were blocked with 2% powdered milk diluted inPBS.

The filters were then incubated with a rat anti-substance P monoclonalantibody (Accurate Chemical & Scientific Corp., Westbury, N.Y.). Ten μlof the antibody was diluted in 10 ml of antibody solution (PBScontaining 20% fetal calf serum and 0.5% Tween-20). The filters wereincubated at room temperature for 1 hour. Unbound antibody was removedwith four 5-minute washes with PBS.

The filters were then incubated with a biotin-conjugated rabbit anti-ratperoxidase antibody (Cappel Laboratories, Melvern, Pa.). The conjugatedantibody was diluted 1:1000 in 10 ml of antibody solution for 2 hours atroom temperature. Excess conjugated antibody was removed with four5-minute washes with PBS.

The filters were pre-incubated for 30 minutes at room temperature with asolution containing 50 μl Vectastain Reagent A (Vector Laboratories) and50 μl Vectastain Reagent B (Vector Laboratories) in 10 ml of antibodysolution that had been allowed to incubate for 30 minutes before use.Excess Vectastain reagents were removed by four 5-minute washes withPBS.

During the preceding wash step, the substrate reagent was prepared. 60mg of horseradish peroxidase reagent (Bio-Rad) was dissolved in 25 ml ofHPLC grade methanol. Approximately 100 ml of PBS and 200 μl H₂ O₂ wereadded just before use. The filters were incubated with the substratereagent for 10 to 20 minutes. The substrate was removed by a vigorouswashing distilled water.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be evident that certain changes and modificationsmay be practiced within the scope of the appended claims.

We claim:
 1. A method for producing a secreted, biologically active,dimerized polypeptide fusion, comprising:introducing into a eukaryotichost cell a first DNA construct comprising a transcriptional promoteroperatively linked to a first secretory signal sequence followeddownstream by and in proper reading frame with a first DNA sequenceencoding a first polypeptide chain of a non-immunoglobulin polypeptidedimer requiring dimerization for biological activity joined to a DNAsequence encoding an immunoglobulin light chain constant region;introducing into said eukaryotic host cell a second DNA constructcomprising a transcriptional promoter operably linked to a secondsecretory signal sequence followed downstream by and in proper readingframe with a second DNA sequence encoding a second polypeptide chain ofsaid non-immunoglobulin polypeptide dimer joined to a DNA sequenceencoding at least one immunoglobulin heavy chain constant region domainselected from the group consisting of C_(H) 1, C_(H) 2, C_(H) 3 andC_(H) 4; growing said host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a dimerizedpolypeptide fusion comprising said first polypeptide chain of anon-immunoglobulin polypeptide dimer joined to said immunoglobulin lightchain constant region and said second polypeptide chain of anon-immunoglobulin polypeptide dimer joined to at least oneimmunoglobulin heavy chain constant region domain, wherein saiddimerized polypeptide fusion exhibits biological activity characteristicof said non-immunoglobulin polypeptide dimer; and isolating saidbiologically active, dimerized polypeptide fusion from said host cell.